Thiotungstate analogues and uses thereof

ABSTRACT

The current invention provides novel thiotungstate derivatives, methods of making novel thiotungstate derivatives, pharmaceutical compositions of novel thiotungstate derivatives, methods of using novel thiotungstate derivatives to treat diseases associated with aberrant vascularization, copper metabolism disorders and obesity and methods of using pharmaceutical compositions of thiotungstate derivatives to treat diseases associated with aberrant vascularization, copper metabolism disorders, neurodegenerative disorders, obesity or NF-κB dysregulation.

1. FIELD

The present invention relates generally to tetrathiotungstatederivatives, methods of making novel tetrathiotungstate derivatives,pharmaceutical compositions of novel tetrathiotungstate derivatives,methods of using novel tetrathiotungstate derivatives and pharmaceuticalcompositions of tetrathiotungstate derivatives to treat or preventdiseases associated with aberrant vascularization, copper metabolismdisorders, neurodegenerative disorders and obesity.

2. BACKGROUND

Most forms of cancer are derived from solid tumors (Shockley et al.,Ann. N.Y. Acad. Sci. 1991, 617: 367-382, which have proven resistant inthe clinic to therapies such as the use of monoclonal antibodies andimmunotoxins. Anti-angiogenic therapy for the treatment of cancer wasdeveloped from the recognition that solid tumors require angiogenesis(i.e., new blood vessel formation) for sustained growth (Folkman, Ann.Surg. 1972, 175: 409-416; Folkman, Mol. Med. 1995, 1(2): 120-122;Folkman, Breast Cancer Res. Treat. 1995, 36(2): 109-118; Hanahan et al.,Cell 1996, 86(3): 353-364). Efficacy of anti-angiogenic therapy inanimal models has been demonstrated (Millauer et al., Cancer Res. 1996,56:1615-1620; Borgstrom et al., Prostrate 1998, 35:1-10; Benjamin etal., J. Clin. Invest. 1999, 103: 159-165; Merajver et al., Proceedingsof Special AACR Conference on Angiogenesis and Cancer 1998, Abstract#B-11, January 22-24). In the absence of angiogenesis, internal celllayers of solid tumors are inadequately nourished. Further, angiogenesis(i.e., aberrant vascularization) has been implicated in numerous otherdiseases (e.g., ocular neovascular disease, macular degeneration,rheumatoid arthritis, etc.). More recently, angiogenesis inhibition hasbeen directly correlated with adipose tissue loss and weight loss inanimal models, which suggests anti-angiogenic therapy may be useful inprevention of obesity (Rupnick et al., Proc. Natl. Acad. Sci. 2002,99:10730-10735).

Contrastingly, normal tissue does not require angiogenesis except underspecialized circumstances (e.g., wound repair, proliferation of theinternal lining of the uterus during the menstrual cycle, etc.).Accordingly, a requirement for angiogenesis is a significant differencebetween tumor cells and normal tissue. Importantly, the dependency oftumor cells on angiogenesis, when compared to normal cells, isquantitatively greater than differences in cell replication and celldeath, between normal tissue and tumor tissue, which are often exploitedin cancer therapy.

Angiogenesis requires copper, as has been shown by numerous studies(Parke et al., Am. J. Pathol. 1988, 137:173-178; Raju et al., Natl.Cancer Inst. 1982, 69: 1183-1188; Ziche et al., Natl. Cancer Inst. 1982,69: 475-482; Gullino, Anticancer Res. 1986, 6(2): 153-158). Attempts atpreventing angiogenesis and hence tumor growth in animal models byreducing in vivo amounts of copper have been reported in the art (Bremet al., Neurosurgery 1990, 26:391-396; Brem et al., Am. J. Pathol. 1990,137(5): 1121-1142; Yoshida et al., Neurosurgery 1995 37(2): 287-295).These approaches incorporated both copper chelators and low copperdiets. More recently, Brewer et al., International Application No.PCT/US99/20374 have shown that the copper chelators, (e.g.,tetrathiomolybdate) may be effective in treating diseases (e.g., solidtumor growth), which require angiogenesis.

In addition to the induction of angiogenesis, copper may also have adirect role in tumor cell growth and survival. High copper levels existin both the plasma and in tumor tissue from patients with many differentsolid cancers (Chakravarty et al., J Cancer Res. Clin. Oncol. 1984, 108:312-315). Recently, tetrathiomolybdate has been shown to down-regulatethe expression of NF-κB as well as inhibit its translocation to thenucleus in the inflammatory breast cancer cell line SUM 149 (Pan et al.,Cancer Res. 2002, 62: 4854-4859). The NF-κB system may be involved inmediating tumor cell survival and thus its down-regulation in tumorcells by tetrathiomolybdate suggests a direct effect of copper chelationon tumor survival.

Accordingly, novel compounds such as tetrathiotungstate compounds, whichare copper chelators, are required to fully explore the potential ofcopper chelators in treating and/or preventing angiogenesis and in tumorcell viability. Such novel tetrathiotungstate compounds may be effectivein treating various diseases associated with angiogenesis such as cancerand obesity along with copper metabolism disorders neurodegenerativedisorders, obesity as well as treating diseases where the NF-κB pathwayis dysregulated such as inflammatory disorders.

3. SUMMARY

The present invention satisfies this and other needs by providing noveltetrathiotungstate derivatives, methods of making noveltetrathiotungstate derivatives, pharmaceutical compositions of noveltetrathiotungstate derivatives, methods of using noveltetrathiotungstate derivatives to treat diseases associated withaberrant vascularization, copper metabolism disorders, neurodegenerativedisorders and obesity and methods of using pharmaceutical compositionsof tetrathiotungstate derivatives to treat or prevent diseasesassociated with aberrant vascularization, copper metabolism disorders,neurodegenerative disorders, obesity or NF-κB dysregulation.

In a first aspect, the present invention provides a compound ofstructural formula (I):

or a solvate, hydrate or N-oxide thereof wherein:R¹, R², R³, R⁵, R⁶ and R⁷ are independently hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heteroalkyl or substituted heteroalkyl;R⁴ and R⁸ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl, substitutedheteroarylalkyl, heteroalkyl or substituted heteroalkyl or are absentwhen N is part of an aromatic ring;optionally, R¹ and R² taken together are alkyldiyl, substitutedalkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;optionally, R⁵ and R⁶ taken together are alkyldiyl, substitutedalkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;optionally, R¹ and R² taken together, R² and R³ taken together and R²and R⁴ taken together are alkyldiyl, substituted alkyldiyl,heteroalkyldiyl or substituted heteroalkyldiyl;optionally, R⁵ and R⁶ taken together, R⁶ and R⁷ taken together and R⁶and R⁸ taken together are alkyldiyl, substituted alkyldiyl,heteroalkyldiyl or substituted heteroalkyldiyl;optionally, R³ and R⁷ taken together are alkyldiyl, substitutedalkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl; andY⁻² is (WS₄)⁻², (W₂S₁₂)⁻², (W₂S₉)⁻², (W₂S₇)⁻², (W₂S₈)⁻², (W₂S₁₁)⁻²,(W₂S₆)⁻² or (W₂S₁₃)⁻².

In a second aspect, the present invention provides pharmaceuticalcompositions of compounds of the invention. The pharmaceuticalcompositions generally comprise one or more compounds of the invention,hydrates or solvates thereof and a pharmaceutically acceptable diluent,carrier, excipient and adjuvant. The choice of diluent, carrier,excipient and adjuvant will depend upon, among other factors, thedesired mode of administration.

In a third aspect, the present invention provides methods for treatingor preventing diseases or disorders characterized by aberrantvascularization, copper metabolism disorders, neurodegenerativedisorders, obesity or NF-κB dysregulation. The methods generally involveadministering to a patient in need of such treatment or prevention atherapeutically effective amount of a compound and/or pharmaceuticalcomposition of the invention.

In a fourth aspect, the current invention provides pharmaceuticalcompositions for treating or preventing diseases or disorderscharacterized by aberrant vascularization, copper metabolism disordersneurodegenerative disorders, obesity or NF-κB dysregulation in a patientin need of such treatment or prevention.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates inhibition of angiogenesis by ammoniumtetrathiotungstate in Matrigel® plug assay.

5. DETAILED DESCRIPTION 5.1 Definitions

“Compounds” refers to compounds encompassed by structural formula (I)disclosed herein and includes any specific compounds within that genericformula whose structure is disclosed herein. Compounds may be identifiedeither by chemical structure and/or chemical name. When the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the identity of the compound. Compounds may contain oneor more chiral centers and/or double bonds and therefore, may exist asstereoisomers, such as double-bond isomers (i.e., geometric isomers),enantiomers or diastereomers. Accordingly, the chemical structuresdepicted herein encompass all possible enantiomers and stereoisomers ofthe illustrated compounds including the stereoisomerically pure form(e.g., geometrically pure, enantiomerically pure or diastereomericallypure) and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures can be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. Compounds may also existin several tautomeric forms including the enol form, the keto form andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.Compounds also include isotopically labeled compounds where one or moreatoms have an atomic mass different from the atomic mass conventionallyfound in nature. Examples of isotopes that may be incorporated into thecompounds of the invention include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O and ³⁵S. Compounds may exist in unsolvated forms aswell as solvated forms, including hydrated forms and as N-oxides. Ingeneral, the hydrated, solvated forms and N-oxides are within the scopeof the present invention. Certain compounds may exist in multiplecrystalline or amorphous forms. In general, all physical forms areequivalent for the uses contemplated herein. Further, it should beunderstood, when partial structures of the compounds are illustrated,that brackets indicate the point of attachment of the partial structureto the rest of the molecule.

“Alkyl” by itself or as part of another substituent, refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane, alkene or alkyne. Typical alkylgroups include, but are not limited to, methyl; ethyls such as ethanyl,ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl,cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. Preferably, an alkyl group comprisesfrom 1 to 20 carbon atoms, more preferably, from 1 to 10 carbon atoms,most preferably, from 1 to 6 carbon atoms.

“Alkanyl” by itself or as part of another substituent, refers to asaturated branched, straight-chain or cyclic alkyl radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Alkyldiyl” by itself or as part of another substituent, refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group derived by the removal of one hydrogen atom from eachof two different carbon atoms of a parent alkane, alkene or alkyne, orby the removal of two hydrogen atoms from a single carbon atom of aparent alkane, alkene or alkyne. The two monovalent radical centers oreach valency of the divalent radical center can form bonds with the sameor different atoms. Typical alkyldiyl groups include, but are notlimited to methandiyl; ethyldiyls such as ethan-1,1-diyl,ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such aspropan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl,cyclopropan-1,1-diyl, cyclopropan-1,2-diyl, prop-1-en-1,1-diyl,prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl,cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used.Preferably, the alkyldiyl group is (C₁-C₂₀) alkyldiyl, more preferably,(C₁-C₁₀) alkyldiyl, most preferably, (C₁-C₆) alkyldiyl. Preferred aresaturated acyclic alkanyldiyl groups in which the radical centers are atthe terminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl(ethano); propan-1,3-diyl (propano); butan-1,4-diyl (butano); and thelike (also referred to as alkyleno, defined infra).

“Alkyleno” by itself or as part of another substituent, refers to astraight-chain alkyldiyl group having two terminal monovalent radicalcenters derived by the removal of one hydrogen atom from each of the twoterminal carbon atoms of straight-chain parent alkane, alkene or alkyne.Typical alkyleno groups include, but are not limited to, methano;ethylenos such as ethano, etheno, ethyno; propylenos such as propano,prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenos such as butano,but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno, but[2]yno,but[1,3]diyno, etc.; and the like. Where specific levels of saturationare intended, the nomenclature alkano, alkeno and/or alkyno is used.Preferably, the alkyleno group is (C₁-C₂₀) alkyleno, more preferably,(C₁-C₁₀) alkyleno, most preferably, (C₁-C₆) alkyleno. Preferred arestraight-chain saturated alkano groups, e.g., methano, ethano, propano,butano, and the like.

“Acyl” by itself or as part of another substituent, refers to a radical—C(O)R³⁰, where R³⁰ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as definedherein. Representative examples include, but are not limited to formyl,acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl,benzylcarbonyl and the like.

“Acylamino” by itself or as part of another substituent, refers to aradical —NR³¹C(O)R³², where R³¹ and R³² are each independently hydrogen,alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl,heteroaryl, heteroarylalkyl, as defined herein. Representative examplesinclude, but are not limited to, formylamino, acetylamino,cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino,benzylcarbonylamino and the like.

“Alkoxy” by itself or as part of another substituent, refers to aradical —OR³³ where R³³ represents an alkyl or cycloalkyl group asdefined herein. Representative examples include, but are not limited to,methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

“Alkoxycarbonyl” by itself or as part of another substituent, refers toa radical —C(O)OR³³ where R³³ is as defined above.

“Aryl” by itself or as part of another substituent, refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Typical aryl groups include, but are not limited to, groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexylene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene and the like. Preferably, an aryl groupcomprises from 6 to 20 carbon atoms, more preferably, from 6 to 12carbon atoms.

“Arylalkyl” by itself or as part of another substituent, refers to anacyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. Preferably, an arylalkyl group is (C₆-C₃₀)arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkylgroup is (C₁-C₁₀) and the aryl moiety is (C₆-C₂₀), more preferably, anarylalkyl group is (C₆-C₂₀) arylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the arylalkyl group is (C₁-C₈) and the aryl moiety is(C₆-C₁₂).

“Cycloalkyl” by itself or as part of another substituent, refers to asaturated or unsaturated cyclic alkyl radical. Where a specific level ofsaturation is intended, the nomenclature “cycloalkanyl” or“cycloalkenyl” is used. Typical cycloalkyl groups include, but are notlimited to, groups derived from cyclopropane, cyclobutane, cyclopentane,cyclohexane and the like. Preferably, the cycloalkyl group is (C₃-C₁₀)cycloalkyl, more preferably, (C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” by itself or as part of another substituent, refersto a saturated or unsaturated cyclic alkyl radical in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atom(s) include, but are not limited to, N, P, O, S,Si, etc. Where a specific level of saturation is intended, thenomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl” is used.Typical cycloheteroalkyl groups include, but are not limited to, groupsderived from epoxides, azirines, thiiranes, imidazolidine, morpholine,piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine and thelike.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkanyl,Heteroalkyldiyl and Heteroalkyleno” by themselves or as part of anothersubstituent, refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl andalkyleno groups, respectively, in which one or more of the carbon atoms(and any associated hydrogen atoms) are each independently replaced withthe same or different heteroatomic groups. Typical heteroatomic groupswhich can be included in these groups include, but are not limited to,—O—, —S—, —O—O—, —S—S—, —O—S—, —NR³⁵R³⁶—, ═N—N═, —N═N—, —N═N—NR³⁷R³⁸,—PR³⁹—, —P(O)₂—, —POR³⁹—, —O—P(O)₂—, —SO—, —SO₂—, —SnR⁴¹R⁴²— and thelike, where R³⁵, R³⁶, R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹ and R⁴² are independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substitutedheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl orsubstituted heteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. Preferably, the heteroaryl group is from 5-20 membered heteroaryl,more preferably from 5-10 membered heteroaryl. Preferred heteroarylgroups are those derived from thiophene, pyrrole, benzothiophene,benzofuran, indole, pyridine, quinoline, imidazole, oxazole andpyrazine.

“Heteroarylalkyl” by itself or as part of another substituent, refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheterorylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-10 membered and theheteroaryl moiety is a 5-20-membered heteroaryl, more preferably, 6-20membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moietyof the heteroarylalkyl is 1-8 membered and the heteroaryl moiety is a5-12-membered heteroaryl.

“Parent Aromatic Ring System” by itself or as part of anothersubstituent, refers to an unsaturated cyclic or polycyclic ring systemhaving a conjugated π electron system. Specifically included within thedefinition of “parent aromatic ring system” are fused ring systems inwhich one or more of the rings are aromatic and one or more of the ringsare saturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Typical parent aromatic ring systems include,but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like.

“Parent Heteroaromatic Ring System” by itself or as part of anothersubstituent, refers to a parent aromatic ring system in which one ormore carbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atoms include, but are not limited to, N, P, O, S,Si, etc. Specifically included within the definition of “parentheteroaromatic ring systems” are fused ring systems in which one or moreof the rings are aromatic and one or more of the rings are saturated orunsaturated, such as, for example, arsindole, benzodioxan, benzofuran,chromane, chromene, indole, indoline, xanthene, etc. Typical parentheteroaromatic ring systems include, but are not limited to, arsindole,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Pharmaceutical composition” refers to at least one compound of theinvention and a pharmaceutically acceptable vehicle, with which thecompound is administered to a patient.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention, which possesses the desired pharmacological activity of theparent compound. Such salts include: (1) acid addition salts, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids such as acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, andthe like; or (2) salts formed when an acidic proton present in theparent compound is replaced by a metal ion, e.g., an alkali metal ion,an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base such as ethanolamine, diethanolamine, triethanolamine,N-methylglucamine and the like.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Patient” includes humans. The terms “human” and “patient” are usedinterchangeably herein.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease).

“Prodrug” refers to a derivative of a drug molecule that requires atransformation within the body to release the active drug. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the parent drug. A hydroxyl containing drug may beconverted to, for example, to a sulfonate, ester or carbonate prodrug,which may be hydrolyzed in vivo to provide the hydroxyl compound. Anamino containing drug may be converted, for example, to a carbamate,amide, enamine, imine, N-phosphonyl, N-phosphoryl or N-sulfenyl prodrug,which may be hydrolyzed in vivo to provide the amino compound. Acarboxylic acid drug may be converted to an ester (including silylesters and thioesters), amide or hydrazide prodrug, which be hydrolyzedin vivo to provide the carboxylic acid compound. Prodrugs for drugswhich functional groups different than those listed above are well knownto the skilled artisan.

“Promoiety” refers to a form of protecting group that when used to maska functional group within a drug molecule converts the drug into aprodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Protecting group” refers to a grouping of atoms that when attached to areactive functional group in a molecule masks, reduces or preventsreactivity of the functional group. Examples of protecting groups can befound in Green et al., “Protective Groups in Organic Chemistry”, (Wiley,2^(nd) ed. 1991) and Harrison et al., “Compendium of Synthetic OrganicMethods”, Vols. 1-8 (John Wiley and Sons, 1971-1996). Representativeamino protecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxy protecting groups include,but are not limited to, those where the hydroxy group is either acylatedor alkylated such as benzyl, and trityl ethers as well as alkyl ethers,tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, -M, —R⁶⁰, —O⁻, ═O,—OR⁶⁰, —SR⁶⁰, —S⁻, ═S, —NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO,—NO₂, ═N₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R⁶⁰, —OS(O₂)O⁻, —OS(O)₂R⁶⁰,—P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰,—C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹,NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹, —NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and—C(NR⁶²)NR⁶⁰R⁶¹ where M is independently a halogen; R⁶⁰, R⁶¹, R⁶² andR⁶³ are independently hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring.Preferably, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O₂)O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹,more preferably, -M, —R⁶⁰, ═O, —OR⁶⁰, —NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂,—S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰,—C(O)NR⁶⁰R⁶¹, —C(O)O⁻, most preferably, -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to ameliorating at least one physical parameter, which may not bediscernible by the patient. In yet another embodiment, “treating” or“treatment” refers to inhibiting the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers to delayingthe onset of the disease or disorder.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the compound, the disease and itsseverity and the age, weight, etc., of the patient to be treated.

Reference will now be made in detail to preferred embodiments of theinvention. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that it is not intended tolimit the invention to those preferred embodiments. To the contrary, itis intended to cover alternatives, modifications, and equivalents as maybe included within the spirit and scope of the invention as defined bythe appended claims.

5.2 Compounds of Structural Formula (I)

In a first embodiment, the compounds of the invention include compoundof structural formula (I):

or a solvate or hydrate or N-oxide thereof wherein:

-   -   R¹, R², R³, R⁵, R⁶ and R⁷ are independently hydrogen, alkyl,        substituted alkyl, aryl, substituted aryl, arylalkyl,        substituted arylalkyl, cycloalkyl, substituted cycloalkyl,        cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl,        substituted heteroaryl, heteroarylalkyl, substituted        heteroarylalkyl, heteroalkyl or substituted heteroalkyl;    -   R⁴ and R⁸ are independently hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, arylalkyl, substituted arylalkyl,        cycloalkyl, substituted cycloalkyl, cycloheteroalkyl,        substituted cycloheteroalkyl, heteroaryl, substituted        heteroaryl, heteroarylalkyl, substituted heteroarylalkyl,        heteroalkyl or substituted heteroalkyl or are absent when N is        part of an aromatic ring;    -   optionally, R¹ and R² taken together are alkyldiyl, substituted        alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;    -   optionally, R⁵ and R⁶ taken together are alkyldiyl, substituted        alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;    -   optionally, R¹ and R² taken together, R² and R³ taken together        and R² and R⁴ taken together are alkyldiyl, substituted        alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;    -   optionally, R⁵ and R⁶ taken together, R⁶ and R⁷ taken together        and R⁶ and R⁸ taken together are alkyldiyl, substituted        alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;    -   optionally, R³ and R⁷ taken together are alkyldiyl, substituted        alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl; and    -   Y² is (WS₄)⁻², (W₂S₁₂)⁻², (W₂S₉)⁻², (W₂S₇)⁻², (W₂S₈)⁻²,        (W₂S₁₁)⁻², (W₂S₆)⁻² or (W₂S₁₃)⁻².

In some embodiment, Y is (WS₄)⁻² and all of R¹, R², R³, R⁴, R⁵, R⁶, R⁷and R⁸ are not hydrogen. In other embodiments, Y is (WS₄)⁻² and all ofR¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are not alkyl.

In still other embodiments,

Preferably, Y is (WS₄)⁻².

In some embodiments, at least one of R¹, R², R³ and R⁴ is not alkyl. Inother embodiments, R¹, R² and R⁴ are hydrogen, alkanyl or substitutedalkanyl. Preferably, R¹, R² and R⁴ are hydrogen, methyl or ethyl.

In still other embodiments, R¹ and R² are alkanyl. Preferably, R¹ and R²are methyl or ethyl.

In still other embodiments, R¹ is alkanyl, substituted alkanyl, alkenyl,substituted alkenyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, cycloalkyl or substituted cycloalkyl. Preferably, R¹ and R²taken together are alkyleno, substituted alkyleno, heteroalkyleno orsubstituted heteroalkyleno. More preferably, R¹ and R² taken togetherare alkyleno or heteroalkyleno.

In still other embodiments, R¹ and R² taken together, R² and R³ takentogether and R² and R⁴ taken together are alkyleno, substitutedalkyleno, heteroalkyleno or substituted heteroalkyleno. Preferably, R¹and R² taken together, R² and R³ taken together and R² and R⁴ takentogether are alkyleno. Preferably, R¹(R²)(R³)(R⁴)N has the structure:

In still other embodiments, R³ and R⁷ taken together are alkyleno,substituted alkyleno, heteroalkyleno or substituted heteroalkyleno.Preferably, R³ and R⁷ taken together are alkyleno or heteroalkyleno.

In still other embodiments, R¹, R² and R⁴ are hydrogen, alkanyl orsubstituted alkanyl and R³ is alkyl, substituted alkyl, alkenyl, aryl,arylalkyl, cycloalkyl or R³ and R⁷ taken together are alkyleno,substituted alkyleno, heteroalkyleno or substituted heteroalkyleno.Preferably, R¹, R² and R⁴ are methyl or ethyl and R³ is alkyl,substituted alkyl, alkenyl, aryl, arylalkyl, cycloalkyl or R³ and R⁷taken together are alkyleno or heteroalkyleno. Preferably, R¹, R² and R⁴are methyl or ethyl and R³ is alkyl, substituted alkyl, alkenyl, aryl,arylalkyl or cycloalkyl.

In still other embodiments, R¹(R²)(R³)(R⁴)N is

In still other embodiments, R¹(R²)(R³)(R⁴)N is

In still other embodiments, R¹(R²)(R³)(R⁴)N is

In still other embodiments, R¹, R² and R⁴ are methyl or ethyl and R³ andR⁷ taken together are alkyleno or heteroalkyleno. Preferably,R¹(R²)(R³)(R⁴)N has the structure:

In still other embodiments, R¹, R² and R⁴ are hydrogen and R³ issubstituted alkyl, cycloalkyl or substituted heteroaryl or R³ and R⁷taken together are alkyleno. In still another embodiment, R¹ and R² arealkanyl and R³ and R⁴ are alkyl, substituted alkyl, aryl, arylalkyl oralkyleno. Preferably, R¹ and R² are methyl or ethyl and R³ and R⁴ arealkyl, substituted alkyl, aryl, arylalkyl or alkyleno.

In still other embodiments, R¹(R²)(R³)(R⁴)N are

wherein R⁹ is a mixture of straight chain alkanyl groups which have atleast eight carbon atoms and not more than eighteen carbon atoms.

In still other embodiments, R¹, R² and R⁴ are hydrogen and R³ issubstituted alkyl, substituted heteroaryl, cycloalkyl or alkyleno.Preferably, R¹(R²)(R³)(R⁴)N has the structure:

In still other embodiments, R¹ and R² taken together are alkyleno,substituted alkyleno, heteroalkyleno or substituted heteroalkyleno, R³is alkyl or substituted alkyl and R⁴ is hydrogen or is absent.Preferably, R¹(R²)(R³)N or R¹(R²)(R³)(R⁴)N has the structure:

5.3 Synthesis of Compounds

The compounds described herein may be obtained via conventionalsynthetic methods illustrated in Schemes 1 and 2. Starting materialsuseful for preparing compounds described herein and intermediatesthereof are commercially available or can be prepared by well-knownsynthetic methods. Substituted ammonium salts (e.g., ammonium hydroxideand ammonium halides) may be either purchased from commercial sources ormay be readily synthesized using well-known synthetic methods (Harrisonet al., “Compendium of Synthetic Organic Methods”, Vols. 1-8 (John Wileyand Sons, 1971-1996); “Beilstein Handbook of Organic Chemistry,”Beilstein Institute of Organic Chemistry, Frankfurt, Germany; Feiser etal., “Reagents for Organic Synthesis,” Volumes 1-17, Wiley Interscience;Trost et al., “Comprehensive Organic Synthesis,” Pergamon Press, 1991;“Theilheimer's Synthetic Methods of Organic Chemistry,” Volumes 1-45,Karger, 1991; March, “Advanced Organic Chemistry,” Wiley Interscience,1991; Larock “Comprehensive Organic Transformations,” VCH Publishers,1989; Paquette, “Encyclopedia of Reagents for Organic Synthesis,” JohnWiley & Sons, 1995). Other methods for synthesis of the compoundsdescribed herein and/or starting materials are either described in theart or will be readily apparent to the skilled artisan. Accordingly, themethods presented in Schemes 1 and 2 herein are illustrative rather thancomprehensive.

As shown above, in Scheme 1, addition of a quaternary ammonium hydroxideto thiotungstate in the presence of water leads to cation exchange(equilibrium to product is driven by removal of volatile ammonia) toprovide the desired thiotungstate derivative.

As shown above, in Scheme 2, addition of a quaternary ammonium halide tothiotungstate in the presence of acetonitrile leads to cation exchange(equilibrium to product is driven by formation of ammonium halide) toprovide the desired thiotungstate derivative.

It should be noted that thiotungstate derivatives where the ammoniumcounterions are different may be prepared from compounds 3 by treatingwith one equivalent of a different ammonium counterion. Such a reactionwould be expected to produce a statistical mixture of products.

Further, those of skill in the art will appreciate that conventionalmethods comprising treating tungstate with an ammonium salt and hydrogensulfide may be used to synthesize many of the compounds describedherein.

5.4 Assays

Those of skill in the art will appreciate that the in vitro and in vivoassays useful for measuring the activity of the compounds describedherein are illustrative rather than comprehensive.

5.4.1 Assay for Endothelial Cell Migration

For endothelial cell migration, transwells are coated with type Icollagen (50 μg/mL) by adding 200 μL of the collagen solution pertranswell, then incubating overnight at 37° C. The transwells areassembled in a 24-well plate and a chemoattractant (e.g., FGF-2) isadded to the bottom chamber in a total volume of 0.8 mL media.Endothelial cells such as human umbilical vein endothelial cells(“HUVEC”), which have been detached from monolayer culture usingtrypsin, are diluted to a final concentration of about 10⁶ cells/mL withserum-free media and 0.2 mL of this cell suspension is added to theupper chamber of each transwell. Inhibitors are added to both the upperand lower chambers, and the migration is allowed to proceed for 5 hrs ina humidified atmosphere at 37° C. The transwells are removed from theplate stained using DiffQuik®. Cells which did not migrate are removedfrom the upper chamber by scraping with a cotton swab and the membranesare detached, mounted on slides, and counted under a high-power field(400×) to determine the number of cells migrated.

5.4.2 Biological Assay of Anti-Invasive Activity

Compounds and/or compositions are tested for their anti-invasivecapacity. The ability of cells such as endothelial cells or tumor cells(e.g., PC-3 human prostatic carcinoma) cells to invade through areconstituted basement membrane (Matrigel®) in an assay known as aMatrigel® invasion assay (Kleinman et al., Biochemistry 1986, 25:312-318; Parish et al., Int. J. Cancer 1992, 52:378-383). Matrigel® is areconstituted basement membrane containing type IV collagen, laminin,heparan sulfate proteoglycans such as perlecan, which bind to andlocalize bFGF, vitronectin as well as transforming growth factor-β(TGFβ, urokinase-type plasminogen activator (uPA), tissue plasminogenactivator (tPA), and the serpin known as plasminogen activator inhibitortype 1 (PAI-1)) (Chambers et al., Canc. Res. 1995, 55:1578-1585).Results obtained in this assay for compounds which target extracellularreceptors or enzymes are typically predictive of the efficacy of thesecompounds in vivo (Rabbani et al., 1995, Int. J. Cancer 63: 840-845).

Such assays employ transwell tissue culture inserts. Invasive cells aredefined as cells which are able to traverse through the Matrigel® andupper aspect of a polycarbonate membrane and adhere to the bottom of themembrane. Transwells (Costar) containing polycarbonate membranes (8.0 μmpore size) are coated with Matrigel® (Collaborative Research), which hasbeen diluted in sterile PBS to a final concentration of 75 μg/mL (60 μLof diluted Matrigel® per insert), and placed in the wells of a 24-wellplate. The membranes are dried overnight in a biological safety cabinet,then rehydrated by adding 100 μL of DMEM containing antibiotics for 1hour on a shaker table. The DMEM is removed from each insert byaspiration and 0.8 mL of DMEM/10% FBS/antibiotics is added to each wellof the 24-well plate such that it surrounds the outside of the transwell(“lower chamber”). Fresh DMEM/antibiotics (100 μL), humanGlu-plasminogen (5 μg/mL), and any compounds to be tested are added tothe top, inside of the transwell (“upper chamber”). The cells which areto be tested are trypsinized and resuspended in DMEM/antibiotics, thenadded to the top chamber of the transwell at a final concentration of800,000 cells/mL. The final volume of the upper chamber is adjusted to200 μL. The assembled plate is then incubated in a humid 5% CO₂atmosphere for 72 hours. After incubation, the cells are fixed andstained using DiffQuik® (Giemsa stain) and the upper chamber is thenscraped using a cotton swab to remove the Matrigel® and any cells whichdid not invade through the membrane. The membranes are detached from thetranswell using an X-acto® blade, mounted on slides using Permount® andcover-slips, then counted under a high-powered (400×) field. An averageof the cells invaded is determined from 5-10 fields counted and plottedas a function of inhibitor concentration.

5.4.3 Tube-Formation Assays of Anti-Angiogenic Activity

Compounds may be tested for anti-angiogenic activity in one of twodifferent assay systems in vitro.

Endothelial cells, for example, human umbilical vein endothelial cells(“HUVEC”) or human microvascular endothelial cells (“HMVEC”) which canbe prepared or obtained commercially, are mixed at a concentration of2×10⁵ cells/mL with fibrinogen (5 mg/mL in phosphate buffered saline(“PBS”) in a 1:1 (v/v) ratio. Thrombin is added (5 units/mL finalconcentration) and the mixture is immediately transferred to a 24-wellplate (0.5 mL per well). The fibrin gel is allowed to form and then VEGFand bFGF are added to the wells (each at 5 ng/mL final concentration)along with the test compound. The cells are incubated at 37° C. in 5%CO₂ for 4 days at which time the cells in each well are counted andclassified as either rounded, elongated with no branches, elongated withone branch, or elongated with 2 or more branches. Results are expressedas the average of 5 different wells for each concentration of compound.Typically, in the presence of angiogenic inhibitors, cells remain eitherrounded or form undifferentiated tubes (e.g., 0 or 1 branch). This assayis recognized in the art to be predictive of angiogenic (oranti-angiogenic) efficacy in vivo (Min et al., Cancer Res. 1996, 56:2428-2433).

In an alternate assay, endothelial cell tube formation is observed whenendothelial cells are cultured on Matrigel® (Schnaper et al., J. Cell.Physiol. 1995, 165:107-118). Endothelial cells (1×10⁴ cells/well) aretransferred onto Matrigel®-coated 24-well plates and tube formation isquantitated after 48 hours. Inhibitors are tested by addition at eitherthe same time as the endothelial cells or at various time pointsthereafter. Tube formation can also be stimulated by adding angiogenicgrowth factors such as bFGF or VEGF, differentiation stimulating agents(e.g., PMA) or combinations thereof.

This assay models angiogenesis by presenting a particular type ofbasement membrane to the endothelial cells, namely the layer of matrix,which migrating and differentiating endothelial cells might be expectedto first encounter. In addition, the matrix components found inMatrigel® (and in basement membranes in situ) or proteolytic productsthereof may also be stimulatory for endothelial cell tube formationwhich makes this model complementary to the fibrin gel angiogenesismodel previously described (Blood et al., Biochim. Biophys. Acta 1990,1032:89-118; Odedra et al., Pharmac. Ther. 1991, 49:111-124). Thecompounds inhibit endothelial cell tube formation in both assays, whichsuggests that the compounds will also have anti-angiogenic activity.

5.4.4 Assays for Inhibition of Proliferation

The ability of the compounds to inhibit the proliferation of endothelialcells may be determined in a 96-well format. Type I collagen (gelatin)is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL perwell for 30 minutes at room temperature). After washing the plate(3×w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4hours (37° C./5% CO₂) in Endothelial Growth Medium (EGM; Clonetics) orM199 media containing 0.1-2% FBS. The media and any unattached cells areremoved at the end of 4 hours and fresh media containing bFGF (1-10ng/mL) or VEGF (1-10 ng/mL) is added to each well. Compounds to betested are added last and the plate is allowed to incubate (37° C./5%CO₂) for 24-48 hrs. MTS (Promega) is added to each well and allowed toincubate from 1-4 hrs. The absorbance at 490 nm, which is proportionalto the cell number, is then measured to determine the differences inproliferation between control wells and those containing test compounds.A similar assay system can be set up with cultured adherent tumor cells.However, collagen may be omitted in this format. Tumor cells (e.g.,3,000-10,000/well) are plated and allowed to attach overnight. Serumfree medium is then added to the wells and the cells are synchronizedfor 24 hrs. Medium containing 10% FBS is then added to each well tostimulate proliferation. Compounds to be tested are included in some ofthe wells. After 24 hrs, MTS is added to the plate and the assaydeveloped and read as described above. A similar methodology may also beemployed to evaluate the effects of the compounds of the invention onthe proliferation of other cell types including tumor cells except thatVEGF and bFGF would not be used to stimulate growth of the cells. Ifthere is evidence of anti-proliferative activity, induction of apoptosismay be measured using TumorTACS (Genzyme).

5.4.5 Assays of Cytotoxicity

The cytotoxic effects of compounds described herein may be determinedfor various cell types including tumor cells, endothelial cells,fibroblasts and macrophages.

A typical assay involves plating cells at a density of 5-10,000 cellsper well in a 96-well plate. Compounds are then added at a variety ofconcentrations and allowed to incubate with the cells for 24 hours. Thecells are washed 3× with media. For cytotoxicity assays (measuring celllysis), a Promega 96-well cytotoxicity kit is used.

5.4.6 Corneal Angiogenesis Model

The protocol used is essentially identical to that described by Volpertet al., J. Clin. Invest. 1996, 98:671-679. Briefly, female Fischer rats(120-140 gms) are anesthetized and pellets (5 μl) comprised of Hydron®,bFGF (150 nM) and the compounds to be tested are implanted into tinyincisions made in the cornea 1.0-1.5 mm from the limbus.Neovascularization is assessed at 5 and 7 days after implantation. Onday 7, animals are anesthetized and infused with a dye such as colloidalcarbon to stain the vessels. The animals are then euthanized, thecorneas fixed with formalin, and the corneas flattened and photographedto assess the degree of neovascularization. Neovessels may bequantitated by imaging the total vessel area or length or simply bycounting vessels.

5.4.7 Matrigel® Plug Assay

This assay is performed essentially as described by Passaniti et al.,Lab Invest. 1992, 67:519-528. Ice-cold Matrigel® (e.g., 500 μL)(Collaborative Biomedical Products, Inc., Bedford, Mass.) is mixed withheparin (e.g., 50 μg/ml), FGF-2 (e.g., 400 ng/ml) and the compound to betested. In some assays, bFGF may be substituted with tumor cells as theangiogenic stimulus. The Matrigel® mixture is injected subcutaneouslyinto 4-8 week-old athymic nude mice at sites near the abdominal midline,preferably 3 injections per mouse. The injected Matrigel® forms apalpable solid gel. Injection sites are chosen such that each animalreceives a positive control plug (such as FGF-2+heparin), a negativecontrol plug (e.g., buffer+heparin) and a plug that includes thecompound being tested for its effect on angiogenesis (e.g.,FGF-2+heparin+compound). All treatments are preferably run intriplicate. Animals are sacrificed by cervical dislocation at about 7days post injection or another time that may be optimal for observingangiogenesis. The mouse skin is detached along the abdominal midline,and the Matrigel® plugs are recovered and scanned immediately at highresolution. Plugs are then dispersed in water and incubated at 37° C.overnight. Hemoglobin (Hb) levels are determined using Drabkin'ssolution (e.g., obtained from Sigma) according to the manufacturers'instructions. The amount of Hb in the plug is an indirect measure ofangiogenesis as it reflects the amount of blood in the sample. Inaddition, or alternatively, animals may be injected prior to sacrificewith a 0.1 ml buffer (preferably PBS) containing a high molecular weightdextran to which is conjugated a fluorophore. The amount of fluorescencein the dispersed plug which is determined fluorimetrically serves as ameasure of angiogenesis in the plug. Staining with mAb anti-CD31 (CD31is platelet-endothelial cell adhesion molecule or “PECAM”) may also beused to confirm neovessel formation and microvessel density in theplugs.

5.4.8 Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay

This assay is performed essentially as described by Nguyen et al.,Microvascular Res. 1994, 47:31-40. A mesh containing either angiogenicfactors (bFGF) or tumor cells plus inhibitors is placed onto the CAM ofan 8-day old chick embryo and the CAM observed for 3-9 days afterimplantation of the sample. Angiogenesis is quantitated by determiningthe percentage of squares in the mesh which contain blood vessels.

5.4.9 In Vivo Assessment Angiogenesis Inhibition and Anti-Tumor EffectsUsing the Matrigel® Plug Assay with Tumor Cells

In this assay, tumor cells, for example, 1-5×10⁶ cells of the 3LL Lewislung carcinoma or the rat prostate cell line MatLyLu, are mixed withMatrigel® and then injected into the flank of a mouse following theprotocol described in section 4.4.7 above. A mass of tumor cells and apowerful angiogenic response can be observed in the plugs after about 5to 7 days. The anti-tumor and anti-angiogenic action of a compound in anactual tumor environment can be evaluated by including it in the plug.Measurement is then made of tumor weight, Hb levels or fluorescencelevels (of a dextran-fluorophore conjugate injected prior to sacrifice).To measure Hb or fluorescence, the plugs are first homogenized with atissue homogenizer.

5.4.10 Xenograft Model of Subcutaneous Tumor Growth

Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma)and Matrigel® (1×10⁶ cells in 0.2 mL) subcutaneously in the right flankof the animals. The tumors are staged to 200 mm³ and then treatment witha test compound is initiated. Tumor volumes are obtained every other dayand the animals are sacrificed after 2 weeks of treatment. The tumorsare excised, weighed and paraffin embedded. Histological sections of thetumors are analyzed by H and E, anti-CD31, Ki-67, TUNEL, and CD68staining.

Other human tumor cell lines including but not limited to PC-3, CWR22R,SK-OV-3, A2780, A549, HCT116, HT29 may also be used to test theanti-tumor activity of the compounds described herein in a similarmanner.

5.4.11 Xenograft Model of Metastasis

The compounds may also be tested for inhibition of late metastasis usingan experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci.USA 1993, 90 5021-5025). Late metastasis involves the steps ofattachment and extravasation of tumor cells, local invasion, seeding,proliferation and angiogenesis. Human prostatic carcinoma cells (PC-3)transfected with a reporter gene, preferably the green fluorescentprotein (GFP) gene, but as an alternative with a gene encoding theenzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ,are inoculated into nude mice. This approach permits utilization ofeither of these markers (fluorescence detection of GFP or histochemicalcolorimetric detection of enzymatic activity) to follow the fate ofthese cells. Cells are injected, preferably, i.v., and metastasesidentified after about 14 days, particularly in the lungs but also inregional lymph nodes, femurs and brain. This mimics the organ tropism ofnaturally occurring metastases in human prostate cancer. For example,GFP-expressing PC-3 cells (1×10⁶ cells per mouse) are injected i.v. intothe tail veins of nude (nu/nu) mice. Animals are treated with a testcomposition at 100 μg/animal/day given q.d. IP. Single metastatic cellsand foci are visualized and quantitated by fluorescence microscopy orlight microscopic histochemistry or by grinding the tissue andquantitative colorimetric assay of the detectable label.

5.4.12 Inhibition of Spontaneous Metastasis In Vivo by HPRG andFunctional Derivatives

The rat syngeneic breast cancer system (Xing et al., Int. J. Cancer1996, 67:423-429) employs Mat BIII rat breast cancer cells. Tumor cells,for example about 10⁶ suspended in 0.1 mL PBS, are inoculated into themammary fat pads of female Fisher rats. At the time of inoculation, a14-day Alza osmotic mini-pump is implanted intraperitoneally to dispensethe test compound. The compound is dissolved in PBS (e.g., 200 mMstock), sterile filtered and placed in the minipump to achieve a releaserate of about 4 mg/kg/day. Control animals receive vehicle (PBS) aloneor a vehicle control peptide in the mini-pump. Animals are sacrificed atabout day 14.

Other models of experimental metastasis may also be used to evaluate thecompounds described herein. These models would utilize the human tumorcell lines described, supra, injected through the tail vein of a nudemouse. Typically, these mice are sacrificed 28 days after tumor cellinoculation and their lungs evaluated for the presence of metastases.

5.4.13 3LL Lewis Lung Carcinoma: Primary Tumor Growth

This tumor line arose spontaneously in 1951 as carcinoma of the lung ina C57BL/6 mouse (Cancer Res. 1955, 15:39. See, also Malave et al., J.Nat'l. Canc. Inst. 1979, 62:83-88). It is propagated by passage inC57BL/6 mice by subcutaneous inoculation and is tested in semiallogeneicC57BL/6×DBA/2 F₁ mice or in allogeneic C3H mice. Typically six animalsper group for subcutaneously implant, or ten for intramuscular implantare used. Tumor may be implanted by subcutaneous inoculation as a 2-4 mmfragment or intramuscularly implanted or subcutaneous implanted as aninoculum of suspended cells of about 0.5-2×10⁶-cells. Treatment begins24 hours after implant or is delayed until a tumor of specified size(usually approximately 400 mg) can be palpated. The test compound isadministered i.p. daily for 11 days

Animals are followed by weighing, palpation, and measurement of tumorsize. Typical tumor weight in untreated control recipients on day 12after intramuscular inoculation is 500-2500 mg. Typical median survivaltime is 18-28 days. A positive control compound, for example,cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used.Results computed include mean animal weight, tumor size, tumor weightand survival time. For confirmed therapeutic activity, the testcomposition should be tested in two multi-dose assays.

5.4.14 3LL Lewis Lung Carcinoma: Primary Growth and SpontaneousMetastasis Model

This model has been utilized by a number of investigators (See, forexample, Gorelik et al., 1980, J. Nat'l. Canc. Inst. 65:1257-1264;Gorelik et al., Rec. Results Canc. Res. 1980, 75:20-28; Isakov et al.,Invasion Metas. 1982, 2:12-32; Talmadge et al., J. Nat'l. Canc. Inst.1982, 69:975-980; Hilgard et al., Br. J. Cancer 1977, 35:78-86). Testmice are male C57BL/6 mice, 2-3 months old. Following subcutaneous,intramuscular or intra-footpad implantation, this tumor producesmetastases, preferentially in the lungs. With some lines of the tumor,the primary tumor exerts anti-metastatic effects and must first beexcised before study of the metastatic phase (see also, O'Reilly et al.,U.S. Pat. No. 5,639,725).

Single-cell suspensions are prepared from solid tumors by treatingminced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3times with PBS (pH 7.4) and suspended in PBS. Viability of the 3LL cellsprepared in this way is generally about 95-99% (by trypan blue dyeexclusion). Viable tumor cells (3×10⁴-5×10⁶) suspended in 0.05 ml PBSare injected subcutaneously, either in the dorsal region or into onehind foot pad of C57BL/6 mice. Visible tumors appear after 3-4 daysafter dorsal sc injection of 10⁶ cells. The day of tumor appearance andthe diameters of established tumors are measured by caliper every twodays.

The treatment is given as one or two doses of compound, per week. Inanother embodiment, the compound is delivered by osmotic minipump.

In experiments involving tumor excision of dorsal tumors, when tumorsreach about 1500 mm³ in size, mice are randomized into two groups: (1)primary tumor is completely excised; or (2) sham surgery is performedand the tumor is left intact. Although tumors from 500-3000 mm³ inhibitgrowth of metastases, 1500 mm³ is the largest size primary tumor thatcan be safely resected with high survival and without local regrowth.After 21 days, all mice are sacrificed and autopsied.

Lungs are removed and weighed. Lungs are fixed in Bouin's solution andthe number of visible metastases is recorded. The diameters of themetastases are also measured using a binocular stereoscope equipped witha micrometer-containing ocular under 8× magnification. On the basis ofthe recorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur et al., J.Lab. Clin. Med. 1977, 89:217-228). Ten days following tumor amputation,25 μg of fluorodeoxyuridine is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice). After 30 min, miceare given 1 μCi of ¹²⁵IdUrd (iododeoxyuridine). One day later, lungs andspleens are removed and weighed and a degree of ¹²⁵IdUrd incorporationis measured using a gamma counter.

In mice with footpad tumors, when tumors reach about 8-10 mm indiameter, mice are randomized into two groups: (1) legs with tumors areamputated after ligation above the knee joints; or (2) mice are leftintact as nonamputated tumor-bearing controls. (Amputation of atumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery). Mice are killed 10-14 days after amputation. Metastases areevaluated as described above.

5.5 Therapeutic Uses

A compound of structural formula (I) and/or a pharmaceutical compositionthereof is administered to a patient, preferably a human, suffering froma disease characterized by aberrant vascularization. Aberrantvascularization includes abnormal neovascularization such as theformation of new blood vessels, larger blood vessels, more branchedblood vessels and any other mechanism, which leads to an increased bloodcarrying capacity to a diseased tissue or site. The compounds andpharmaceutical compositions thereof may be used to treat and/or preventaberrant vascularization.

Preferably, diseases characterized by aberrant vascularization include,but are not limited to, cancer (e.g., any vascularized tumor,preferably, a solid tumor, including but not limited to, carcinomas ofthe lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes,liver, parotid, bilary tract, colon, rectum, cervix, uterus,endometrium, kidney, bladder, prostrate, thyroid, squamous cellcarcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, sarcomas (e.g., angiosarcomas, chondrosarcomas)),arthritis, diabetes, arteriosclerosis, arteriovenous, malformations,corneal graft neovascularization, delayed wound healing, diabeticretinopathy, age related macular degeneration, granulations, burns,hemophilic joints, rheumatoid arthritis, hypertrophic scars, neovascularglaucoma, nonunion fractures, Osier Weber Syndrome, psoriasis,granuloma, retrolental fibroplasia, pterygium, scleroderma, trachoma,vascular adhesions, ocular neovascularization, parasitic diseases,hypertrophy following surgery, inhibition of hair growth, maculardegeneration (including both wet and dry type), rheumatoid arthritis andosteoarthritis. Diseases characterized by aberrant vascularization whichare preferably treated and/or prevented by administration of a compoundof structural formula (I) and/or a pharmaceutical composition thereofinclude cancer, macular degeneration and rheumatoid arthritis.

Further, a compound of structural formula (I) and/or a pharmaceuticalcomposition thereof may be administered to a patient, preferably ahuman, suffering from a disease associated with copper metabolismdisorders (e.g., Wilson's disease) to treat and/or prevent such adisease.

Still further, a compound of structural formula (I) and/or apharmaceutical composition thereof may be administered to a patient,preferably a human, to treat and/or prevent obesity. The compounds ofstructural formula (I) may be also used to reduce levels of inflammatorycytokines (e.g., TNF-α, TNF-β, IL-8, etc.) and plasminogen activatorinhibitor, which may be associated with angiogenesis and obesity(Loskutoff et al., Ann. N.Y. Acad. Sci., 2000, 902:272-281; Pan et al.,Cancer Res., 2002, 62:4854-4859; Hanada et al., Cytokine Growth FactorRev. 2002, 13:413-421; Chen et al., Science 2002, 296:1634-5; Miyake etal., J. Neuropathol. Exp. Neurol. 59:18-28; Koch et al., Science 1992,258:1798-801; Osawa et al., Infect. Immun. 2002, 70:6294-6301; Bajou etal., Nat. Med. 1998, 4:923-8).

Still further, a compound of structural formula (I) and/or apharmaceutical composition thereof may be administered to a patient,preferably a human, suffering from a neurodegenerative disorder, totreat and/or prevent the neurodegenerative disorder. Elevated levels ofcopper have been reported in the art to mediate the pathobiology ofvarious neurodegenerative disorders including Alzheimer's disease,amyotrophic lateral sclerosis (ALS) and prion disease (Llanos et al.,DNA Cell Biol. 2002, 21: 259-270; Carri et al., Funct. Neurol 2001,16:181-188; Perry et al., CNS Drugs 2002, 16:339-352; Kowalik-Jankowskaet al., Environ Health Perspect, 2002, 5: 869-870; Maynard et al., J.Biol. Chem. 2002, 277, 44670-44676; Gnjec et al., Front Biosci. 2002,16-23; Strausak et al., Brain Res. Bull. 2001, 55: 175-185; Brown, BrainRes. Bull. 2001, 55:165-173; Brown, Biochem. Soc. Trans 2002,30:742-745).

Still further, in accordance with the invention, a compound ofstructural formula (I) and/or a pharmaceutical composition thereof maybe administered to a patient, preferably a human, to treat diseasescharacterized by dysregulated activity of the NF-κB or dysregulatedinflammation of inflammatory response.

Further, in certain embodiments, a compounds and/or pharmaceuticalcompositions thereof are administered to a patient, preferably, a human,as a preventative measure against various diseases or disorderscharacterized by aberrant vascularization, copper metabolism disorders,neurodegenerative disorders, obesity or NF-κB dysregulation.Accordingly, compounds of structural Formula (I) and/or pharmaceuticalcompositions thereof may be used for the prevention of one disease ordisorder and concurrently treating another (e.g., preventing Wilson'sdisease or Alzheimer's while treating cancer).

5.6 Therapeutic/Prophylactic Administration

The compounds of structural Formula (I) and/or pharmaceuticalcompositions thereof may be advantageously used in human medicine. Aspreviously described in Section 4.5, supra, compounds of structuralFormula (I) and/or pharmaceutical compositions thereof are useful forthe treatment and/or prevention of various diseases or disorderscharacterized by aberrant vascularization, copper metabolism disorders,neurodegenerative disorders, obesity or NF-κB dysregulation.

When used to treat and/or prevent the above disease or disorders,compounds of structural Formula (I) and/or pharmaceutical compositionsmay be administered or applied singly, or in combination with otheragents. The compounds of structural Formula (I) and/or pharmaceuticalcompositions thereof may also be administered or applied singly, incombination with other pharmaceutically active agents (e.g., otheranti-cancer agents, other anti-angiogenic agents, other chelators suchas zinc, penicillamine, etc. and other anti-obesity agents), includingother compounds of structural Formula (I) and/or pharmaceuticalcompositions thereof.

Methods of treatment and prophylaxis by administration to a patient of atherapeutically effective amount of a compound of structural Formula (I)and/or pharmaceutical composition thereof are provided herein. Thepatient may be an animal, more preferably, a mammal and most preferably,a human.

The present compounds of structural Formula (I) and/or pharmaceuticalcompositions thereof, are preferably administered orally. The compoundsof structural Formula (I) and/or pharmaceutical compositions thereof mayalso be administered by any other convenient route, for example, byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.). Administration can be systemic or local. Various delivery systemsare known, (e.g., encapsulation in liposomes, microparticles,microcapsules, capsules, etc.) that can be used to administer a compoundof structural Formula (I) and/or pharmaceutical composition thereof.Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically, particularly to theears, nose, eyes, or skin. The preferred mode of administration is leftto the discretion of the practitioner and will depend in-part upon thesite of the medical condition. In most instances, administration willresult in the release of the compounds of structural Formula (I) and/orpharmaceutical compositions thereof into the bloodstream.

In specific embodiments, it may be desirable to administer one or morecompounds of structural Formula (I) and/or pharmaceutical compositionthereof locally to the area in need of treatment. This may be achieved,for example, and not by way of limitation, by local infusion duringsurgery, topical application, e.g., in conjunction with a wound dressingafter surgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. In one embodiment, administration can beby direct injection at the site (or former site) of aberrantvascularization (e.g., cancer or arthritis).

In certain embodiments, it may be desirable to introduce one or morecompounds of structural Formula (I) and/or pharmaceutical compositionsthereof into the central nervous system by any suitable route, includingintraventricular, intrathecal and epidural injection. Intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

A compound of structural Formula (I) and/or pharmaceutical compositionthereof may also be administered directly to the lung by inhalation. Foradministration by inhalation, a compound of structural Formula (I)and/or pharmaceutical composition thereof may be conveniently deliveredto the lung by a number of different devices. For example, a MeteredDose Inhaler (“MDI”), which utilizes canisters that contain a suitablelow boiling propellant, (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or anyother suitable gas) may be used to deliver compounds of structuralFormula (I) and/or pharmaceutical compositions thereof directly to thelung.

Alternatively, a Dry Powder Inhaler (“DPI”) device may be used toadminister a compound of structural Formula (I) and/or pharmaceuticalthereof to the lung. DPI devices typically use a mechanism such as aburst of gas to create a cloud of dry powder inside a container, whichmay then be inhaled by the patient. DPI devices are also well known inthe art. A popular variation is the multiple dose DPI (“MDDPI”) system,which allows for the delivery of more than one therapeutic dose. Forexample, capsules and cartridges of gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of a compound ofstructural Formula (I) and a suitable powder base such as lactose orstarch for these systems.

Another type of device that may be used to deliver a compound ofstructural Formula (I) and/or pharmaceutical composition thereof to thelung is a liquid spray device supplied, for example, by AradigmCorporation, Hayward, Calif. Liquid spray systems use extremely smallnozzle holes to aerosolize liquid drug formulations that may then bedirectly inhaled into the lung.

In one embodiment, a nebulizer is used to deliver a compound ofstructural Formula (I) and/or pharmaceutical composition thereof to thelung. Nebulizers create aerosols from liquid drug formulations by using,for example, ultrasonic energy to form fine particles that may bereadily inhaled (see e.g., Verschoyle et al., British J. Cancer, 1999,80, Suppl. 2, 96). Examples of nebulizers include devices supplied bySheffield Pharmaceuticals, Inc (See, Armer et al., U.S. Pat. No.5,954,047; van der Linden et al., U.S. Pat. No. 5,950,619; van derLinden et al., U.S. Pat. No. 5,970,974), and Batelle PulmonaryTherapeutics, Columbus, Ohio.

In another embodiment, an electrohydrodynamic (“EHD”) aerosol device isused to deliver a compound of structural Formula (I) and/orpharmaceutical composition thereof to the lung. EHD aerosol devices useelectrical energy to aerosolize liquid drug solutions or suspensions(see e.g., Noakes et al., U.S. Pat. No. 4,765,539). The electrochemicalproperties of the formulation may be important parameters to optimizewhen delivering a compound of structural Formula (I) and/orpharmaceutical composition thereof to the lung with an EHD aerosoldevice and such optimization is routinely performed by one of skill inthe art. EHD aerosol devices may more efficiently deliver compounds tothe lung than other pulmonary delivery technologies.

In some embodiments, the compounds of structural Formula (I) and/orpharmaceutical compositions thereof can be delivered in a vesicle, inparticular a liposome (See, Langer, 1990, Science 249:1527-1533; Treatet al., in “Liposomes in the Therapy of Infectious Disease and Cancer,”Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);see generally “Liposomes in the Therapy of Infectious Disease andCancer,” Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989)).

In other embodiments, the compounds of structural Formula (I) and/orpharmaceutical compositions thereof can be delivered via sustainedrelease systems, preferably oral sustained release systems. In oneembodiment, a pump may be used (See, Langer, supra, Sefton, 1987, CRCCrit. Ref Biomed Eng. 14:201; Saudek et al., 1989, N. Engl. J Med.321:574).

In yet other embodiments, polymeric materials can be used (see “MedicalApplications of Controlled Release,” Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,” Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Langer et al., 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; seealso Levy et al., 1985, Science 228: 190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In stillother embodiments, polymeric materials are used for oral sustainedrelease delivery. Preferred polymers include sodiumcarboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose and hydroxyethylcellulose (most preferred,hydroxypropyl methylcellulose). Other preferred cellulose ethers havebeen described (Alderman, Int. J. Pharm. Tech. & Prod. Mfr., 1984,5(3)1-9). Factors affecting drug release are well known to the skilledartisan and have been described in the art (Bamba et al., Int. J.Pharm., 1979, 2, 307).

In still other embodiment, enteric-coated preparations can be used fororal sustained release administration. Preferred coating materialsinclude polymers with a pH-dependent solubility (i.e., pH-controlledrelease), polymers with a slow or pH-dependent rate of swelling,dissolution or erosion (i.e., time-controlled release), polymers thatare degraded by enzymes (i.e., enzyme-controlled release) and polymersthat form firm layers that are destroyed by an increase in pressure(i.e., pressure-controlled release).

In still other embodiments, osmotic delivery systems are used for oralsustained release administration (Verma et al., Drug Dev. Ind. Pharm.,2000, 26:695-708). In yet other embodiments, OROS™ osmotic devices areused for oral sustained release delivery devices (Theeuwes et al., U.S.Pat. No. 3,845,770; Theeuwes et al., U.S. Pat. No. 3,916,899).

In still other embodiments, a controlled-release system can be placed inproximity of the target of the compounds and/or pharmaceuticalcomposition of the invention, thus requiring only a fraction of thesystemic dose (See, e.g., Goodson, in “Medical Applications ofControlled Release,” supra, vol. 2, pp. 115-138 (1984). Othercontrolled-release systems discussed in Langer, 1990, Science249:1527-1533 may also be used.

5.7 Pharmaceutical Compositions

The present pharmaceutical compositions contain a therapeuticallyeffective amount of one or more compounds of structural Formula (I),preferably in purified form, together with a suitable amount of apharmaceutically acceptable vehicle, so as to provide the form forproper administration to a patient. When administered to a patient, thecompounds of structural Formula (I) and pharmaceutically acceptablevehicles are preferably sterile. Water is a preferred vehicle when thecompound of structural Formula (I) is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid vehicles, particularly for injectable solutions.Suitable pharmaceutical vehicles also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present pharmaceutical compositions, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. In addition, auxiliary, stabilizing, thickening, lubricating andcoloring agents may be used.

Pharmaceutical compositions comprising a compound of structural Formula(I) may be manufactured by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries, whichfacilitate processing of compounds of structural Formula (I) intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

The present pharmaceutical compositions can take the form of solutions,suspensions, emulsion, tablets, pills, pellets, capsules, capsulescontaining liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. In one embodiment, the pharmaceuticallyacceptable vehicle is a capsule (see e.g., Grosswald et al., U.S. Pat.No. 5,698,155). Other examples of suitable pharmaceutical vehicles havebeen described in the art (see Remington: The Science and Practice ofPharmacy, Philadelphia College of Pharmacy and Science, 20^(th) Edition,2000).

For topical administration a compound of structural Formula (I) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asis well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration. Systemic formulationsmay be made in combination with a further active agent that improvesmucociliary clearance of airway mucus or reduces mucous viscosity. Theseactive agents include, but are not limited to, sodium channel blockers,antibiotics, N-acetyl cysteine, homocysteine and phospholipids.

In some embodiments, the compounds of structural Formula (I) areformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to human beings.Typically, compounds of structural Formula (I) for intravenousadministration are solutions in sterile isotonic aqueous buffer. Forinjection, a compound of structural Formula (I) may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. When necessary, the pharmaceuticalcompositions may also include a solubilizing agent. Pharmaceuticalcompositions for intravenous administration may optionally include alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. When thecompound of structural Formula (I) is administered by infusion, it canbe dispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. When the compound of structuralFormula (I) is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Pharmaceutical compositions for oral delivery may be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered pharmaceutical compositions may contain one or moreoptionally agents, for example, sweetening agents such as fructose,aspartame or saccharin; flavoring agents such as peppermint, oil ofwintergreen, or cherry coloring agents and preserving agents, to providea pharmaceutically palatable preparation. Moreover, where in tablet orpill form, the pharmaceutical compositions may be coated to delaydisintegration and absorption in the gastrointestinal tract, therebyproviding a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds ofstructural Formula (I). In these later platforms, fluid from theenvironment surrounding the capsule is imbibed by the driving compound,which swells to displace the agent or agent composition through anaperture. These delivery platforms can provide an essentially zero orderdelivery profile as opposed to the spiked profiles of immediate releaseformulations. A time delay material such as glycerol monostearate orglycerol stearate may also be used. Oral compositions can includestandard vehicles such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Such vehiclesare preferably of pharmaceutical grade.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,saline, alkyleneglycols (e.g., propylene glycol), polyalkylene glycols(e.g., polyethylene glycol) oils, alcohols, slightly acidic buffersbetween pH 4 and pH 6 (e.g., acetate, citrate, ascorbate at betweenabout 5.0 mM to about 50.0 mM) etc. Additionally, flavoring agents,preservatives, coloring agents, bile salts, acylcarnitines and the likemay be added.

For buccal administration, the pharmaceutical compositions may take theform of tablets, lozenges, etc. formulated in conventional manner.

Liquid drug formulations suitable for use with nebulizers and liquidspray devices and EHD aerosol devices will typically include a compoundof structural Formula (I) with a pharmaceutically acceptable vehicle.Preferably, the pharmaceutically acceptable vehicle is a liquid such asalcohol, water, polyethylene glycol or a perfluorocarbon. Optionally,another material may be added to alter the aerosol properties of thesolution or suspension of compounds of the invention. Preferably, thismaterial is liquid such as an alcohol, glycol, polyglycol or a fattyacid. Other methods of formulating liquid drug solutions or suspensionsuitable for use in aerosol devices are known to those of skill in theart (see, e.g., Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat.No. 5,556,611).

A compound of structural Formula (I) may also be formulated in rectal orvaginal pharmaceutical compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, a compound ofstructural Formula (I) may also be formulated as a depot preparation.Such long acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, a compound of structural Formula (I) maybe formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

When a compound of structural Formula (I) is acidic, it may be includedin any of the above-described formulations as the free acid, apharmaceutically acceptable salt, a solvate or hydrate. Pharmaceuticallyacceptable salts substantially retain the activity of the free acid, maybe prepared by reaction with bases and tend to be more soluble inaqueous and other protic solvents than the corresponding free acid form.

5.8 Therapeutic Doses

A compound of structural Formula (I), and/or pharmaceutical compositionthereof, will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent diseases or disorderscharacterized by aberrant vascularization, copper metabolism disorders,neurodegenerative disorders and obesity the compounds of structuralFormula (I) and/or pharmaceutical compositions thereof, are administeredor applied in a therapeutically effective amount.

The amount of a compound of structural Formula (I) that will beeffective in the treatment of a particular disorder or conditiondisclosed herein will depend on the nature of the disorder or conditionand can be determined by standard clinical techniques known in the art,as previously described. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theamount of a compound of structural Formula (I) administered will, ofcourse, be dependent on, among other factors, the subject being treated,the weight of the subject, the severity of the affliction, the manner ofadministration and the judgment of the prescribing physician.

For example, the dosage may be delivered in a pharmaceutical compositionby a single administration, by multiple applications or controlledrelease. In one embodiment, the compounds of structural Formula (I) aredelivered by oral sustained release administration. Preferably, in thisembodiment, the compounds of structural Formula (I) are administeredtwice per day (more preferably, once per day). Dosing may be repeatedintermittently, may be provided alone or in combination with other drugsand may continue as long as required for effective treatment of thedisease state or disorder.

Suitable dosage ranges for oral administration depend on potency, butare generally between about 0.001 mg to about 200 mg of a compound ofstructural Formula (I) per kilogram body weight. Dosage ranges may bereadily determined by methods known to the artisan of ordinary skill theart.

Suitable dosage ranges for intravenous (i.v.) administration are about0.01 mg to about 100 mg per kilogram body weight. Suitable dosage rangesfor intranasal administration are generally about 0.01 mg/kg body weightto about 1 mg/kg body weight. Suppositories generally contain about 0.01milligram to about 50 milligrams of a compound of structural Formula (I)per kilogram body weight and comprise active ingredient in the range ofabout 0.5% to about 10% by weight. Recommended dosages for intradermal,intramuscular, intraperitoneal, subcutaneous, epidural, sublingual orintracerebral administration are in the range of about 0.001 mg to about200 mg per kilogram of body weight. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro or animal model testsystems. Such animal models and systems are well-known in the art.

The compounds of structural Formula (I) are preferably assayed in vitroand in vivo, for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays can be used to determinewhether administration of a specific compound of structural Formula (I)or a combination of compounds of structural Formula (I) is preferred fortreating or preventing diseases or disorders characterized by aberrantvascularization, copper metabolism disorders, neurodegenerativedisorders and obesity. The compounds of structural Formula (I) may alsobe demonstrated to be effective and safe using animal model systems.

Preferably, a therapeutically effective dose of a compound of structuralFormula (I) described herein will provide therapeutic benefit withoutcausing substantial toxicity. Toxicity of compounds of structuralFormula (I) may be determined using standard pharmaceutical proceduresand may be readily ascertained by the skilled artisan. The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Acompound of structural Formula (I) will preferably exhibit particularlyhigh therapeutic indices in treating disease and disorders. The dosageof a compound of structural Formula (I) described herein will preferablybe within a range of circulating concentrations that include aneffective dose with little or no toxicity.

5.9 Combination Therapy

In certain embodiments of the present invention, the compounds ofstructural Formula (I) and/or pharmaceutical compositions thereof can beused in combination therapy with at least one other therapeutic agent orwith radiation therapy. The compound of structural Formula (I) and/orpharmaceutical composition thereof and the other therapeutic agent canact additively or, more preferably, synergistically. In someembodiments, a compound of structural Formula (I) and/or pharmaceuticalcomposition thereof is administered concurrently with the administrationof another therapeutic agent, which may be part of the samepharmaceutical composition as the compound of structural Formula (I) ora different pharmaceutical composition. In other embodiments, apharmaceutical composition of structural Formula (I) is administeredprior or subsequent to administration of another therapeutic agent.

In some embodiments, the compounds of structural Formula (I) and/orpharmaceutical compositions thereof can be used in combination therapywith other chemotherapeutic agents (e.g., alkylating agents (e.g.,nitrogen mustards (e.g., cyclophosphamide, ifosfamide, mechlorethamine,melphalen, chlorambucil, hexamethylmelamine, thiotepa), alkyl sulfonates(e.g., busulfan), nitrosoureas, triazines) antimetabolites (e.g., folicacid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine,cytosine arabinoside, etc.), purine analogs (e.g., mercaptopurine,thiogunaine, pentostatin, etc.), natural products (e.g., vinblastine,vincristine, etoposide, tertiposide, dactinomycin, daunorubicin,doxurubicin, bleomycin, mithrmycin, mitomycin C, L-asparaginase,interferon alpha), platinum coordination complexes (e.g., cis-platinum,carboplatin, etc.), mitoxantrone, hydroxyurea, procarbazine, hormonesand antagonists (e.g., prednisone, hydroxyprogesterone caproate,medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone,flutamide, leuprolide, etc.), anti-angiogenesis agents or inhibitors(e.g., angiostatin, retinoic acids and paclitaxel, estradiolderivatives, thiazolopyrimidine derivatives, etc.), apoptosis-inducingagents (e.g., antisense nucleotides that block oncogenes which inhibitapoptosis, tumor suppressors, TRAIL, TRAIL polypeptide, Fas-associatedfactor 1, interleukin-1β-converting enzyme, phosphotyrosine inhibitors,RXR retinoid receptor agonists, carbostyril derivatives, etc.),chelators (penicillamine, zinc, trientine, etc.) and other anti-obesityagents.

5.10 Therapeutic Kits

Therapeutic kits comprising the compounds of structural Formula (I)and/or pharmaceutical compositions thereof are also provided herein. Thetherapeutic kits may also contain other compounds (e.g.,chemotherapeutic agents, natural products, hormones or antagonists,anti-angiogenesis agents or inhibitors, apoptosis-inducing agents orchelators) and/or pharmaceutical compositions thereof.

Therapeutic kits may have a single container which contains the compoundof structural Formula (I) and/or pharmaceutical compositions thereofwith or without other components (e.g., other compounds and/orpharmaceutical compositions thereof) or may have distinct container foreach component. Preferably, therapeutic kits include a compound ofstructural Formula (I) and/or a pharmaceutical composition thereofpackaged for use in combination with the co-administration of a secondcompound (preferably, a chemotherapeutic agent, a natural product, ahormone or antagonist, a anti-angiogenesis agent or inhibitor, anapoptosis-inducing agent or a chelator) or a pharmaceutical compositionthereof. The components of the kit may be pre-complexed or eachcomponent may be in a separate distinct container prior toadministration to a patient.

The components of the kit may be provided in one or more liquidsolutions, preferably, an aqueous solution, more preferably, a sterileaqueous solution. The components of the kit may also be provided assolids, which may be converted into liquids by addition of suitablesolvents, which are preferably provided in another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid.

Preferably, a therapeutic kit will contain apparatus (e.g., one or moreneedles, syringes, eye droppers, pipette, etc.), which enablesadministration of the components of the kit.

6. EXAMPLES

The invention is further defined by reference to the following examples,which describe in detail, preparation of compounds of the invention andmethods for assaying for biological activity. It will be apparent tothose skilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of theinvention.

6.1 Example 1 General Procedure for Synthesis of TetrathiotungstateDerivatives

The quaternary ammonium hydroxide (2 eq.), as a commercially-availableaqueous solution, was added to ammonium tetrathiotungstate (1 eq.) anddeionized water was added until all the solid material was dissolved.The solution was placed on a rotary evaporator under vacuum (ca. 5-10torr) with the bath at 20° C. for one hour and the water was replaced asneeded to maintain a constant volume. The reaction mixture was thenallowed to evaporate to dryness and the resulting yellow solid wasrecrystallized from deionized water and isopropanol. The solid wascollected by filtration, washed with isopropanol and diethyl ether, andthen dried under high vacuum for 24 hours in a vacuum desiccator in thepresence of P₂O₅.

6.2 Example 2 General Procedure for Synthesis of TetrathiotungstateDerivatives

The quaternary ammonium halide (2 eq.) as a solid was added to asuspension of ammonium tetrathiotungstate (1 eq.) in dry acetonitrile (5mL per mmol of tetrathiotungstate) and the resulting mixture was stirredat room temperature under nitrogen for 18 hours. If this procedureresulted in a precipitate, the solid was collected by filtration, washedwith isopropanol and diethyl ether and was recrystallized from deionizedwater and isopropanol. The yellow crystals were collected by filtration,washed with isopropanol and diethyl ether and dried under high vacuumfor 24 hours in a vacuum desiccator in the presence of P₂O₅. If thesolution remained clear, the solvent was removed in vacuo, the residuewas taken up in dichloromethane, washed three times with water, oncewith brine, dried (Na₂SO₄), and the solution was concentrated. Theresulting oil or solid was dried under high vacuum for 24 hours in avacuum desiccator in the presence of P₂O₅.

6.3 Example 3 General Procedure for Synthesis of TetrathiotungstateDerivatives

The quaternary ammonium halide (2 eq.) as a solution in deionized water(10 mL per mmol of tetrathiotungstate) was added to a suspension ofammonium tetrathiotungstate (1 eq.) in dry acetonitrile (20 mL per mmolof tetrathiotungstate) and the resulting mixture was stirred at roomtemperature for 18 hours. If this procedure resulted in a precipitate,the solid was collected by filtration, washed with water, isopropanoland diethyl ether, and then dried under high vacuum for 24 hours in avacuum desiccator in the presence of P₂O₅. If the solution remainedclear, the reaction mixture was first filtered, and the filtrate wasconcentrated in vacuo. The resulting solid was recrystallized fromdeionized water and isopropanol, the yellow crystals collected byfiltration, washed with isopropanol and diethyl ether and then driedunder high vacuum for 24 hours in a vacuum desiccator in the presence ofP₂O₅.

6.4 Example 4 Tetrathiotungstate, bis(choline)

This compound was prepared from ammonium tetrathiotungstate (158 mg,0.454 mmol) and a 50% by weight aqueous solution of choline hydroxide(222 mg, 0.916 mmol) according to the procedure of Example 1, whichafforded 151 mg (64%) of the title compound as bright yellow crystals:IR (KBr, cm⁻¹) 3402, 459; ¹H NMR (300 MHz, DMSO-d₆) δ 5.21 (t, J=4.8 Hz,2H), 3.88-3.81 (m, 4H), 3.46-3.43 (m, 4H), 3.14 (s, 18H); ¹³C NMR (75MHz, DMSO-d₆) δ66.8 (2C), 55.2 (2C), 53.1 (6C); ES MS m/z (choline)⁺104.3; UV (H₂O) 393.5 nm (ε=16730). Anal. Calcd for C₁₀H₂₈N₂O₂S₄W: C,23.08; H, 5.42; N, 5.38; S, 24.65. Found: C, 23.17; H, 5.28; N, 5.43; S,24.87.

6.5 Example 5 Tetrathiotungstate, bis(triethylmethyl ammonium)

This compound was prepared from ammonium tetrathiotungstate (164 mg,0.471 mmol) and a 20% by weight aqueous solution oftriethylmethylammonium hydroxide (627 mg, 0.941 mmol) according to theprocedure of Example 1, which provided 147 mg (61%) of the titlecompound as bright yellow crystals: IR (KBr, cm⁻¹) 460; ¹H NMR (300 MHz,DMSO-d₆) δ 3.29 (q, J=6.9 Hz, 12H), 2.91 (s, 6H), 1.21 (t, J=6.9 Hz,18H); ¹³C NMR (75 MHz, DMSO-d₆) δ 55.0 (6C), 46.0 (2C), 7.5 (6C); ES MSm/z (triethylmethyl ammonium)⁺ 116.4; UV (H₂O) 393.5 nm (ε=16730). Anal.Calcd for C₁₄H₃₆N₂S₄W: C, 30.88; H, 6.66; N, 5.14; S, 23.55. Found: C,30.87; H, 6.33; N, 5.18; S, 23.77.

6.6 Example 6 Tetrathiotungstate, bis(triethylphenyl ammonium)

This compound was prepared from ammonium tetrathiotungstate (155 mg,0.444 mmol) and a 10% by weight aqueous solution oftriethylphenylammonium hydroxide (1.74 g, 0.889 mmol) according to theprocedure of Example 1, which provided 198 mg (69%) of the titlecompound as bright yellow crystals: IR (KBr, cm⁻¹) 455; ¹H NMR (300 MHz,DMSO-d₆) δ 7.92 (d, J=8.4 Hz, 4H), 7.71-7.57 (m, 6H), 3.91 (q, J=7.1 Hz,12H), 1.06 (t, J=7.1 Hz, 18H); ¹³C NMR (75 MHz, DMSO-d₆) δ 141.7 (2C),130.4 (4C), 130.0 (2C), 122.6 (4C), 55.3 (6C), 7.8 (6C); ES MS m/z(triethylphenyl ammonium)⁺ 178.4; UV (H₂O) 393.5 nm (ε=15600). Anal.Calcd for C₂₄H₄₀N₂S₄W: C, 43.11; H, 6.03; N, 4.19; S, 19.18. Found: C,42.99; H, 5.73; N, 4.25; S, 19.31.

6.7 Example 7 Tetrathiotungstate, bis(1,4-dimethylpyridinium)

This compound was prepared from ammonium tetrathiotungstate (163 mg,0.467 mmol) and 1,4-dimethylpyridinium iodide (221 mg, 0.940 mmol)according to the procedure of Example 2, which provided 143 mg (58%) ofthe title compound as bright yellow crystals: IR (KBr, cm⁻¹) 458; ¹H NMR(300 MHz, DMSO-d₆) δ 8.88 (d, J=6.4 Hz, 4H), 7.96 (d, J=6.4 Hz, 4H),4.32 (s, 6H), 2.60 (s, 6H); ¹³C NMR (75 MHz, DMSO-d₆) δ 158.1 (2C),144.8 (4C), 128.0 (4C), 47.1 (2C), 21.4 (2C); ES MS m/z(1,4-dimethylpyridinium)⁺ 108.3; UV (H₂O) 393.5 nm (c=16030). Anal.Calcd for C₁₄H₂₀N₂S₄W: C, 31.82; H, 3.81; N, 5.30; S, 24.27. Found: C,31.67; H, 3.77; N, 5.32; S, 24.13.

6.8 Example 8 Tetrathiotungstate, bis(1,1-dimethylpyrrolidinium)

This compound was prepared from ammonium tetrathiotungstate (300 mg,0.861 mmol) and 1,1-dimethylpyrrolidinium iodide (400 mg, 1.76 mmol)according to the procedure of Example 3, which provided 223 mg (51%) ofthe title compound as bright yellow crystals: IR (KBr, cm⁻¹) 455; ¹H NMR(300 MHz, DMSO-d₆) δ 3.53-3.47 (m, 8H), 3.13 (s, 12H), 2.14-2.08 (m,8H); ¹³C NMR (75 MHz, DMSO-d₆) δ 64.8 (4C), 51.0 (4C), 21.5 (4C); ES MSm/z (1,1-dimethylpyrrolidinium)⁺ 100.3; UV (H₂O) 393.5 nm (ε=16950).Anal. Calcd for C₁₂H₂₈N₂S₄W: C, 28.12; H, 5.51; N, 5.47; S, 25.03.Found: C, 27.90; H, 5.47; N, 5.56; S, 25.01.

6.9 Example 9 Tetrathiotungstate, bis(trimethylphenylammonium)

This compound was prepared from ammonium tetrathiotungstate (167 mg,0.479 mmol) and phenyltrimethyl-ammonium chloride (166 mg, 0.968 mmol)according to the procedure of Example 2, which provided 139 mg (50%) ofthe title compound as bright yellow crystals: IR (KBr, cm⁻¹) 459; ¹H NMR(300 MHz, DMSO-d₆) δ 7.99 (d, J=8.2 Hz, 4H), 7.68-7.55 (m, 6H), 3.64 (s,18H); ¹³C NMR (75 MHz, DMSO-d₆) δ 147.3 (2C), 130.1 (4C), 130.0 (2C),120.5 (4C), 56.4 (6C); ES MS m/z (trimethylphenylammonium)⁺ 136.2; UV(H₂O) 394.0 nm (s=15630). Anal. Calcd for C₁₈H₂₈N₂S₄W: C, 36.99; H,4.83; N, 4.79; S, 21.94. Found: C, 36.88; H, 4.72; N, 4.90; S, 21.92.

6.10 Example 10 Tetrathiotungstate, bis(acetylcholine)

This compound was prepared from ammonium tetrathiotungstate (171 mg,0.491 mmol) and acetylcholine chloride (179 mg, 0.987 mmol) according tothe procedure of Example 2, which provided 163 mg (55%) of the titlecompound as bright yellow crystals: IR (KBr, cm⁻¹) 1749, 1729, 473, 456;¹H NMR (300 MHz, DMSO-d₆) δ 4.47-4.41 (m, 4H), 3.72-3.69 (m, 4H), 3.16(s, 18H), 2.07 (s, 6H); ¹³C NMR (75 MHz, DMSO-d₆) δ 169.9 (2C), 63.8(2C), 57.9 (2C), 53.0 (6C), 20.7 (2C); ES MS m/z (acetylcholine)⁺ 146.4;UV (H₂O) 393.5 nm (ε=15400). Anal. Calcd for C₁₄H₃₂N₂O₄S₄W: C, 27.82; H,5.34; N, 4.63; S, 21.22. Found: C, 27.62; H, 5.12; N, 4.68; S, 20.71.

6.11 Example 11 Tetrathiotungstate,bis[alkyldimethyl(phenylmethyl)ammonium]

This compound was prepared from ammonium tetrathiotungstate (320 mg,0.920 mmol) and benzalkonium chloride (664 mg, 1.84 mmol) according tothe procedure of Example 2, which afforded 651 mg (74%) of the titlecompound as a thick, red oil: IR (film, cm⁻¹) 466; ¹H NMR (300 MHz,DMSO-d₆) δ 7.59-7.48 (m, 10H), 4.56 (s, 4H), 3.31-3.23 (m, 4H), 2.97 (s,12H), 1.84-1.72 (m, 4H), 1.32-1.22 (m, 40H), 0.88-0.82 (m, 6H); ES MSm/z [dodecyldimethyl(phenylmethyl)ammonium]⁺ 304.7,[tetradecyldimethyl(phenylmethyl) ammonium]⁺ 332.7; UV (DMSO) 399.0 nm(ε=10400).

6.12 Example 12 Tetrathiotungstate, suberyldicholine

This compound was prepared from ammonium tetrathiotungstate (299 mg,0.860 mmol) and suberyldicholine diiodide (516 mg, 0.860 mmol) accordingto the procedure of Example 2, which afforded 115 mg (20%) of the titlecompound as bright yellow crystals: IR (KBr, cm⁻¹) 1733, 1719, 455; ¹HNMR (300 MHz, DMSO-d₆) δ 4.48-4.42 (m, 4H), 3.73-3.69 (m, 4H), 3.17 (s,18H), 2.35 (t, J=7.4 Hz, 4H), 1.59-1.48 (m, 4H), 1.32-1.26 (m, 4H); ¹³CNMR (75 MHz, DMSO-d₆) δ 172.3 (2C), 63.7 (2C), 57.7 (2C), 52.8 (6C),33.2 (2C), 28.0 (2C), 23.9 (2C); UV (H₂O) 394.0 nm (ε=15570).

6.13 Example 13 Tetrathiotungstate, pentane-1,5-bis(trimethylammonium)

This compound was prepared from ammonium tetrathiotungstate (140 mg,0.402 mmol) and N,N-pentamethylenebis(trimethylammonium iodide) (195 mg,0.442 mmol) according to the procedure of Example 3, which afforded 109mg (54%) of the title compound as a bright yellow powder: IR (KBr, cm⁻¹)456; ¹H NMR (300 MHz, D₂O) δ 3.34-3.26 (m, 4H), 3.07 (s, 18H), 1.89-1.77(m, 4H), 1.45-1.34 (m, 2H); UV (H₂O) 394.0 nm (s=15950). Anal. Calcd forC₁₁H₂₈N₂S₄W: C, 26.40; H, 5.64; N, 5.60; S, 25.63. Found: C, 26.60; H,5.26; N, 5.75; S, 24.64.

6.14 Example 14 Tetrathiotungstate, butane-1,4-bis(trimethylammonium)

This compound was prepared from ammonium tetrathiotungstate (200 mg,0.574 mmol) and N,N-tetramethylenebis(trimethylammonium iodide) (271 mg,0.632 mmol) according to the procedure of Example 3 and afforded 185 mg(66%) of the title compound as a bright yellow powder: IR (KBr, cm⁻¹)456; ¹H NMR (300 MHz, D₂O) δ 3.45-3.35 (m, 4H), 3.11 (s, 18H), 1.92-1.82(m, 4H); UV (H₂O) 394.0 nm (ε=15990). Anal. Calcd for C₁₀H₂₆N₂S₄W: C,24.69; H, 5.39; N, 5.76; S, 26.37. Found: C, 24.77; H, 5.35; N, 5.85; S,25.80.

6.15 Example 15 Tetrathiotungstate, propane-1,3-bis(trimethylammonium)

This compound was prepared from ammonium tetrathiotungstate (201 mg,0.578 mmol) and N,N-timethylenebis(trimethylammonium iodide) (263 mg,0.635 mmol) according to the procedure of Example 3, and afforded 192 mg(70%) of the title compound as a bright yellow powder: IR (KBr, cm⁻¹)456; UV (H₂O) 393.5 nm (ε=16190). Anal. Calcd for C₉H₂₄N₂S₄W: C, 22.88;H, 5.12; N, 5.93; S, 27.15. Found: C, 22.94; H, 5.01; N, 6.01; S, 26.79.

6.16 Example 15 Tetrathiotungstate, ethylenebis(trimethylammonium)

This compound was prepared from ammonium tetrathiotungstate (200 mg,0.573 mmol) and ethylenebis(trimethylammonium iodide) (249 mg, 0.623mmol) according to the procedure of Example 3, and afforded 171 mg (65%)of the title compound as a bright yellow powder: IR (KBr, cm⁻¹) 459; UV(H₂O) 393.5 nm (ε=15720). Anal. Calcd for C₈H₂₂N₂S₄W: C, 20.96; H, 4.84;N, 6.11; S, 27.98. Found: C, 20.88; H, 4.71; N, 6.21; S, 27.39.

6.17 Example 17 Tetrathiotungstate, bis(N-benzyl-2-phenylethyl ammonium)

This compound was prepared from ammonium tetrathiotungstate (295 mg,0.848 mmol) and N-benzyl-2-phenylethylammonium chloride (422 mg, 1.70mmol) according to the procedure of Example 3, but with the addition of6 mL of deionized water, and afforded 317 mg (51%) of the title compoundas an orange solid: IR (KBr, cm⁻¹) 455; ¹H NMR (300 MHz, DMSO-d₆) δ 8.83(br s, 4H), 7.57-7.52 (m, 4H), 7.48-7.40 (m, 6H), 7.36-7.23 (m, 10H),4.22 (s, 4H), 3.21-3.15 (m, 4H), 3.03-2.95 (m, 4H); ¹³C NMR (75 MHz,DMSO-d₆) δ 137.4 (2C), 132.5 (2C), 130.0 (4C), 128.9 (2C), 128.72 (8C),128.68 (4C), 126.8 (2C), 50.6 (2C), 48.1 (2C), 31.8 (2C); ES MS m/z[N-benzyl-2-phenylethyl ammonium]⁺ 212.4; UV (DMSO) 399.5 nm (ε=16270).

6.18 Example 18 Tetrathiotungstate, bis(1-ethyl-3-methyl-1H-imidazolium)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.400g, 1.15 mmol) and 1-ethyl-3-methyl-1H-imidazolium chloride (0.354 g,2.41 mmol) according to the procedure of Example 3 giving the titlecompound (0.217 g, 35%) as a bright yellow solid: IR (KBr pellet, cm⁻¹)3438, 3068, 1569, 1560, 1169, 450; ¹H NMR (300 MHz, DMSO-d₆) δ 9.22 (s,1H), 7.78 (s, 1 μl), 7.70 (s, 1H), 4.21 (q, 2H, J=7.3 Hz), 3.31 (s, 3μl), 1.42 (t, 3 μl, J=7.3 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 136.4, 123.4,121.8, 44.0, 35.6, 15.1; MS m/z (C₆H₁₁N₂)⁺ 111.3; UV (H₂O) 394 nm(ε=15,891); Anal. calcd for C₁₂H₂₂N₄WS₄: C, 26.97; H, 4.15; N, 10.48; S,24.00. Found: C, 26.91, H, 3.92; N, 10.55; S, 23.67.

6.19 Example 19 Tetrathiotungstate, bis(benzyltrimethylammonium)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.200g, 0.574 mmol) and benzyltrimethylammonium hydroxide (0.48 g of a 40%aqueous solution, 1.15 mmol) according to the procedure of Example 1 toprovide giving the title compound (0.246 g, 70%) as a bright yellowsolid: IR (KBr pellet, cm⁻¹) 3446, 2999, 1456, 458; ¹H NMR (300 MHz,DMSO-d₆) δ 7.53-7.55 (m, 10H), 4.56 (s, 4 H), 3.05 (s, 18H); ¹³C NMR (75MHz, DMSO-d₆) δ 132.7, 130.2, 128.8, 128.3, 67.7, 51.7 (t); MS m/z(C₁₀H₁₆N)⁺ 150.3; UV (H₂O) 394 nm (ε=15,027); Anal. calcd forC₂₀H₃₂N₂WS₄: C, 39.21; H, 5.27; N, 4.57; S, 20.94. Found: C, 39.28, H,4.88; N, 4.65; S, 20.89.

6.20 Example 20 Tetrathiotungstate,bis(2-hydroxyiminomethyl-1-methyl-pyridinium)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.200g, 0.574 mmol) and 2-pyridinealdoxime methochloride (0.198 g, 1.15 mmol)according to the procedure of Example 3 to give the title compound(0.198 g, 59%) as a bright yellow solid: IR (KBr pellet, cm⁻¹) 3077,1508, 1005, 455; ¹H NMR (300 MHz, DMSO-d₆) δ 9.04 (d, 1H, J=5.9 Hz),8.68 (s, 1H), 8.55 (app t, 1H), 8.37 (d, 1 H, J=8.0 Hz), 8.07 (app t,1H), 4.39 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 147.3, 146.7, 144.8,141.7, 127.1, 124.7, 46.1; MS m/z (C₇H₉N₂O)⁺ 137.2; UV (H₂O) 394 nm(ε=15380); Anal. calcd for C₁₄H₁₈N₄O₂WS₄: C, 28.67; H, 3.09; N, 9.55; S,21.87. Found: C, 28.51, H, 2.87; N, 9.63; S, 21.55.

6.21 Example 21 Tetrathiotungstate, bis(acetyl-β-methylcholine)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.200g, 0.574 mmol) and acetyl-β-methylcholine chloride (0.235 g, 1.20 mmol)according to the procedure of Example 3 giving the title compound (0.115g, 32%) as a bright yellow solid: IR (KBr pellet, cm⁻¹) 3452, 3008,1735, 1252, 454; ¹H NMR (300 MHz, DMSO-d₆) δ 5.27 (m, 1H), 3.58-3.74 (m,2H), 3.13 (s, 9H), 2.06 (s, 3H), 1.24 (d, 3H, J=6.3 Hz); ¹³C NMR (75MHz, DMSO-d₆) δ 169.5, 67.6, 65.2, 53.2, 21.1, 18.5; MS m/z (C₈H₁₈NO₂)⁺160.3; UV (H₂O) 394 nm (ε=15831); Anal. calcd for C₁₆H₃₆N₂O₄S₄W: C,30.38; H, 5.74; N, 4.43; S, 20.28. Found: C, 30.10, H, 5.62; N, 4.47; S,20.47.

6.22 Example 22 Tetrathiotungstate, (succinylcholine)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.400g, 1.15 mmol) and succinylcholine chloride dihydrate (0.456 g, 1.15mmol) according to the procedure of Example 3 to give the title compound(0.414 g, 60%) as a bright yellow solid: IR (KBr pellet, cm⁻¹) 3005,1732, 1208, 1150, 455; ¹H NMR (300 MHz, DMSO-d₆) δ 4.47 (m, 4H),3.68-3.72 (m, 4H), 3.30 (d, 18H, J=4.2 Hz), 2.66 (m, 4 H); ¹³C NMR (75MHz, DMSO-d₆) δ 171.3, 58.0, 52.9, 28.4; UV (H₂O) 394 nm (ε=15513);Anal. calcd for C₁₄H₃₀N₂O₄S₄W: C, 27.91; H, 5.02; N, 4.65; S, 21.29.Found: C, 27.84, H, 4.80; N, 4.66; S, 21.06.

6.23 Example 23 Tetrathiotungstate, (ethylene-1,2-bisammonium)

This compound was prepared from tetrathiotungstate, bis(ammonium) (0.300g, 0.862 mmol), ammonium chloride (0.092 g, 1.72 mmol) andethylenediamine (57.6 μl, 0.862 mmol) according to the procedure ofExample 3 to give the title compound (0.257 g, 80%) as a bright yellowsolid: IR (KBr pellet, cm⁻¹) 3002, 1435, 1025, 451; ¹H NMR (300 MHz,DMSO-d₆) δ 7.89 (bs, 6H), 3.09 (s, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ36.8; UV (H₂O) 394 nm (ε=13291); Anal. calcd for C₂H₁₀N₂S₄W: C, 6.42; H,2.69; N, 7.49; S, 34.27. Found: C, 6.58, H, 2.43; N, 7.49; S, 32.98.

6.24 Example 24 Moisture Stability of Tetrathiotungstate Salts

Salts of tetrathiotungstate were placed in an acrylic chamber at roomtemperature with 95% relative humidity for two weeks. The samples wereanalyzed for purity according to the previously reported method with theexception that the absorbance was monitored at 493 nm and the molarabsorptivity was 15710 M⁻¹ cm⁻¹ (McDonald et. al., Inorg. Chim. Acta1983, 72, 205-210). The results are reported below in Table 1

TABLE 1 Name % degradation Tetrathiotungstate, −0.12 bis(triethylphenylammonium) Tetrathiotungstate, −0.46 bis(ammonium) Tetrathiotungstate,−0.46 bis(trimethylphenylammonium) Tetrathiotungstate, 0.41bis(benzyltrimethylammonium) Tetrathiotungstate, 2.4 bis(acetylcholine)Tetrathiotungstate, bis(choline) 3.0 Tetrathiotungstate, bis(1-ethyl-3-1.3 methyl-1H-imidazolium) Tetrathiotungstate, bis(1,4- 2.0dimethylpyridinium) Tetrathiotungstate, bis(acetyl-β- 4.2 methylcholine)Tetrathiotungstate, bis(1,1- 0.04 dimethylpyrrolidinium)Tetrathiotungstate, bis(2- 5.6 hydroxyiminomethyl-1-methyl- pyridinium)Tetrathiotungstate, pentane-1,5- 19.8* bis(trimethylammonium)Tetrathiotungstate, 8.6* ethylenebis(trimethylammonium)Tetrathiotungstate, propane-1,3- 23.8* bis(trimethylammonium)Tetrathiotungstate, butane-1,4- 5.7* bis(trimethylammonium)Tetrathiotungstate, 23.3* (succinylcholine) Tetrathiotungstate, 18.4*suberyldicholine Tetrathiomolybdate, bis 56^(‡) ± 5 (ammonium)Tetrathiomolybdate, bis (choline) 36^(‡) ± 2 *partially soluble^(‡)average of 2 data points

6.25 Example 25 Copper-Binding Ability of Tetrathiotungstate Salts

The copper-binding ability of tetrathiotungstate salts was determinedaccording to the ability of tetrathiotungstate salts to inhibit cysteineautooxidation as reported in Table 2 below.

TABLE 2 Inhibition of Cysteine Autooxidation (100 μM Cys, 100 μMcoumarin-3-carboxylic acid, 100 μM CuSO₄) inhibitor inhibitorconcentration % inhibition trientine 50 μM 39.71% Tetrathiotungstate, 50μM 95.94% bis(ammonium) Tetrathiotungstate, 50 μM 91.32%bis(triethylphenyl ammonium) Tetrathiotungstate, 50 μM 91.53%bis(trimethylphenylammonium) Tetrathiotungstate, 50 μM 94.62%bis(benzyltrimethylammonium) Tetrathiotungstate, 50 μM 90.36%bis(acetylcholine) Tetrathiotungstate, bis(choline) 50 μM 92.54%Tetrathiotungstate, bis(1-ethyl-3- 50 μM 91.16% methyl-1H-imidazolium)Tetrathiotungstate, bis(1,4- 50 μM 89.98% dimethylpyridinium)Tetrathiotungstate, bis(acetyl-β- 50 μM 90.40% methylcholine)Tetrathiotungstate, bis(1,4- 50 μM 89.66% dimethylpyridinium)Tetrathiotungstate, bis(2- 50 μM 91.15% hydroxyiminomethyl-1-methyl-pyridinium) Tetrathiotungstate, pentane-1,5- 50 μM 90.69%bis(trimethylammonium) Tetrathiotungstate, bis(choline) 50 μM 91.99%Tetrathiomolybdate, 50 μM 94.24% bis(ammonium) Tetrathiotungstate, 50 μM89.60% ethylenebis(trimethylammonium) Tetrathiotungstate, propane-1,3-50 μM 92.95% bis(trimethylammonium) Tetrathiotungstate, butane-1,4- 50μM 91.75% bis(trimethylammonium) trientine 10 μM 4.27%Tetrathiomolybdate, 10 μM 87.71% bis(ammonium) Tetrathiotungstate, 10 μM84.66% ethylenebis(trimethylammonium) Tetrathiotungstate, propane-1,3-10 μM 85.53% bis(trimethylammonium) Tetrathiotungstate, butane-1,4- 10μM 87.32% bis(trimethylammonium) trientine 10 μM 17.72%Tetrathiomolybdate, 10 μM 76.69% bis(ammonium) Tetrathiotungstate, 10 μM81.38% (succinylcholine) Tetrathiotungstate, 10 μM 77.83%suberyldicholine trientine  1 μM 0.00% Tetrathiomolybdate,  1 μM 68.48%bis(ammonium) Tetrathiotungstate,  1 μM 66.06% (succinylcholine)Tetrathiotungstate,  1 μM 67.74% suberyldicholine

6.25 Example 25 Inhibition of Angiogenesis in Matrigel® Plug Assay byAmmonium Tetrathiotungstate

Ammonium tetrathiotungstate was assayed in the Matrigel® plug assay asdescribed in Section 5.4.7, supra. Two positive controls were used withpositive control 1 measured five days after implantation when treatmentbegan and negative control 2 measured five days after implantation whentreatment ended. Two negative controls were used with negative control 1measured five days after implantation when treatment began and negativecontrol 2 measured five days after implantation when treatment ended. Ascan be seen in FIG. 1, treatment with ammonium tetrathiotungstateresulted in about 34% inhibition using this assay.

Finally, it should be noted that there are alternative ways ofimplementing the present invention. Accordingly, the present embodimentsare to be considered as illustrative and not restrictive, and theinvention is not to be limited to the details given herein, but may bemodified within the scope and equivalents of the appended claims. Allpublications and patents cited herein are incorporated by reference intheir entirety.

1. A compound of structural formula (I):

or a solvate or hydrate thereof wherein: R¹, R², R³, R⁵, R⁶ and R⁷ areindependently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heteroalkyl or substituted heteroalkyl; R⁴ and R⁸ are independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heteroalkyl orsubstituted heteroalkyl or are absent when N is part of an aromaticring; optionally, R¹ and R² taken together are alkyldiyl, substitutedalkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl; optionally,R⁵ and R⁶ taken together are alkyldiyl, substituted alkyldiyl,heteroalkyldiyl or substituted heteroalkyldiyl; optionally, R¹ and R²taken together, R² and R³ taken together and R² and R⁴ taken togetherare alkyldiyl, substituted alkyldiyl, heteroalkyldiyl or substitutedheteroalkyldiyl; optionally, R⁵ and R⁶ taken together, R⁶ and R⁷ takentogether and R⁶ and R⁸ taken together are alkyldiyl, substitutedalkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl; optionally,R³ and R⁷ taken together are alkyldiyl, substituted alkyldiyl,heteroalkyldiyl or substituted heteroalkyldiyl; and Y⁻² is (W₂S₁₂)⁻²,(W₂S₉)⁻², (W₂S₇)⁻², (W₂S₈)⁻², (W₂S₁₁)⁻², (W₂S₆)⁻² or (W₂S₁₃)⁻².
 2. Acompound of structural formula (I):

or a solvate or hydrate thereof wherein: Y⁻² is (WS₄)⁻²; and a) whereinR¹(R²)(R³)(R⁴)N has the structure:

wherein R¹(R²)(R³)N has the structure:

b) wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R⁹ is n-C₈H₁₇, n-C₉H₁₉, n-C₁₀H₂₁, n-C₁₁H₂₃, n-C₁₂H₂₅, n-C₁₃H₂₇,n-C₁₄H₂₉, n-C₁₅H₃₁, n-C₁₆H₃₃, n-C₁₇H₃₅ or n-C₁₈H₃₇; c) whereinR¹(R²)(R³)(R⁴)N has the structure:

d) wherein R¹(R²)(R³)(R⁴)N has the structure:

e) wherein R¹(R²)(R³)N has the structure:

f) wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R⁵, R⁶ and R⁷ are independently hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heteroalkyl or substituted heteroalkyl; R⁸is independently hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heteroalkyl or substituted heteroalkyl or are absent when N is part ofan aromatic ring; optionally, R⁵ and R⁶ taken together are alkyldiyl,substituted alkyldiyl, heteroalkyldiyl or substituted heteroalkyldiyl;and optionally, R⁵ and R⁶ taken together, R⁶ and R⁷ taken together andR⁶ and R⁸ taken together are alkyldiyl, substituted alkyldiyl,heteroalkyldiyl or substituted heteroalkyldiyl.
 3. The compound of claim2, wherein

a) wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R¹(R²)(R³)N has the structure:

b) wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R⁹ is n-C₈H₁₇, n-C₉H₁₉, n-C₁₀H₂₁, n-C₁₁H₂₃, n-C₁₂H₂₅, n-C₁₃H₂₇,n-C₁₄H₂₉, n-C₁₅H₃₁, n-C₁₆H₃₃, n-C₁₇H₃₅ or n-C₁₈H₃₇; c) whereinR¹(R²)(R³)(R⁴)N has the structure:

d) wherein R¹(R²)(R³)(R⁴)N has the structure:

e) wherein R¹(R²)(R³)N has the structure:

f) wherein R¹(R²)(R³)(R⁴)N has the structure:


4. The compound of claim 2, wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R¹(R²)(R³)N has the structure:


5. The compound of claim 2, wherein R¹(R²)(R³)(R⁴)N has the structure:

wherein R⁹ is n-C₈H₁₇, n-C₉H₁₉, n-C₁₀H₂₁, n-C₁₁H₂₃, n-C₁₂H₂₅, n-C₁₃H₂₇,n-C₁₄H₂₉, n-n-C₁₅H₃₁, n-C₁₆H₃₃, n-C₁₇H₃₅ or n-C₁₈H₃₇.
 6. The compound ofclaim 2, wherein R¹(R²)(R³)(R⁴)N has the structure:


7. The compound of claim 2, wherein R¹(R²)(R³)(R⁴)N has the structure:


8. The compound of claim 2, wherein R¹(R²)(R³)N has the structure:


9. The compound of claim 2, wherein R¹(R²)(R³)N has the structure:

10-35. (canceled)
 36. A pharmaceutical composition comprising thecompound of claim 2 and a pharmaceutically acceptable diluent, excipientor adjuvant.
 37. A method for treating cancer in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the compound of claim
 2. 38. A method for treatingcancer in a patient comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of the pharmaceuticalcomposition of claim
 36. 39. The method of claim 37 further comprisingadministering to the patient in need of such treatment a therapeuticallyeffective amount of another anti-cancer agent or a pharmaceuticalcomposition comprising i) said another anti-cancer agent and ii) apharmaceutically acceptable diluent, excipient or adjuvant.
 40. Themethod of claim 38 further comprising administering to the patient inneed of such treatment a therapeutically effective amount of anotheranti-cancer agent or a pharmaceutical composition comprising i) saidanother anti-cancer agent and ii) a pharmaceutically acceptable diluent,excipient or adjuvant.
 41. The method of claim 37 further comprisingadministering to the patient in need of such treatment a therapeuticallyeffective amount of zinc or a pharmaceutical composition comprising i)zinc and ii) a pharmaceutically acceptable diluent, excipient oradjuvant.
 42. The method of claim 38 further comprising administering tothe patient in need of such treatment a therapeutically effective amountof zinc or a pharmaceutical composition comprising i) zinc and ii) apharmaceutically acceptable diluent, excipient or adjuvant.
 43. Themethod of claim 37, wherein the cancer is breast cancer, renal cancer,brain cancer, colon cancer, prostrate cancer, chondrosarcoma orangiosarcoma.
 44. A method for treating wet type macular degeneration orrheumatoid arthritis in a patient comprising administering to a patientin need of such treatment a therapeutically effective amount of thecompound of claim
 2. 45. A method for treating wet type maculardegeneration or rheumatoid arthritis in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the pharmaceutical composition of claim
 36. 46. Amethod for treating aberrant vascularization in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the compound of claim
 2. 47. A method for treatingaberrant vascularization in a patient comprising administering to apatient in need of such treatment a therapeutically effective amount ofthe pharmaceutical composition of claim
 36. 48. A method for treatingexcess copper levels in a patient comprising administering to a patientin need of such treatment a therapeutically effective amount of thecompound of claim
 2. 49. A method for treating excess copper levels in apatient comprising administering to a patient in need of such treatmenta therapeutically effective amount of the pharmaceutical composition ofclaim
 36. 50. A method for treating obesity in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the compound of claim
 2. 51. A method for treatingobesity in a patient comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of the pharmaceuticalcomposition of claim
 36. 52. The method of claim 50 further comprisingadministering to the patient in need of such treatment a therapeuticallyeffective amount of another anti-obesity agent or a pharmaceuticalcomposition comprising i) said another anti-obesity agent and ii) apharmaceutically acceptable diluent, excipient or adjuvant.
 53. Themethod of claim 51 further comprising administering to the patient inneed of such treatment a therapeutically effective amount of anotheranti-obesity agent or a pharmaceutical composition comprising i) saidanother anti-obesity agent and ii) a pharmaceutically acceptablediluent, excipient or adjuvant.
 54. A method for treatingneurodegenerative disease in a patient comprising administering to apatient in need of such treatment a therapeutically effective amount ofthe compound of claim
 2. 55. A method for treating neurodegenerativedisease in a patient comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of the pharmaceuticalcomposition of claim
 36. 56. The method of claim 54, wherein theneurodegenerative disease is Alzheimer's disease, amyotrophic lateralsclerosis or prion disease.
 57. The method of claim 55, wherein theneurodegenerative disease is Alzheimer's disease, amyotrophic lateralsclerosis or prion disease.
 58. The method of claim 38, wherein thecancer is breast cancer, renal cancer, brain cancer, colon cancer,prostrate cancer, chondrosarcoma or angiosarcoma.
 59. The compound ofclaim 6, wherein R¹(R²)(R³)(R⁴)N has the structure:

and wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N
 60. A pharmaceuticalcomposition comprising the compound of claim 59 and a pharmaceuticallyacceptable diluent, excipient or adjuvant.
 61. The compound of claim 4,wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N or R¹(R²)(R³)N═R⁵(R⁶)(R⁷)N. 62.The compound of claim 5, wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N. 63.The compound of claim 7, wherein R¹(R²)(R³)(R⁴)N has the structure:

and wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N.
 64. The compound of claim8, wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N.
 65. The compound of claim 9,wherein R¹(R²)(R³)(R⁴)N═R⁵(R⁶)(R⁷)(R⁸)N.
 66. A method for treatingcancer in a patient comprising administering to a patient in need ofsuch treatment a therapeutically effective amount of the compound ofclaim
 59. 67. The method of claim 66 further comprising administering tothe patient in need of such treatment a therapeutically effective amountof another anti-cancer agent or a pharmaceutical composition comprisingi) said another anti-cancer agent and ii) a pharmaceutically acceptablediluent, excipient or adjuvant.
 68. The method of claim 66 furthercomprising administering to the patient in need of such treatment atherapeutically effective amount of zinc or a pharmaceutical compositioncomprising i) zinc and ii) a pharmaceutically acceptable diluent,excipient or adjuvant.
 69. The method of claim 66, wherein the cancer isbreast cancer, renal cancer, brain cancer, colon cancer, prostratecancer, chondrosarcoma or angiosarcoma.
 70. A method for treating wettype macular degeneration or rheumatoid arthritis in a patientcomprising administering to a patient in need of such treatment atherapeutically effective amount of the compound of claim
 59. 71. Amethod for treating aberrant vascularization in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the compound of claim
 59. 72. A method for treatingexcess copper levels in a patient comprising administering to a patientin need of such treatment a therapeutically effective amount of thecompound of claim
 59. 73. A method for treating obesity in a patientcomprising administering to a patient in need of such treatment atherapeutically effective amount of the compound of claim
 59. 74. Themethod of claim 73 further comprising administering to the patient inneed of such treatment a therapeutically effective amount of anotheranti-obesity agent or a pharmaceutical composition comprising i) saidanother anti-obesity agent and ii) a pharmaceutically acceptablediluent, excipient or adjuvant.
 75. A method for treatingneurodegenerative disease in a patient comprising administering to apatient in need of such treatment a therapeutically effective amount ofthe compound of claim
 59. 76. The method of claim 75, wherein theneurodegenerative disease is Alzheimer's disease, amyotrophic lateralsclerosis or prion disease.
 77. A method for treating cancer, aberrantvascularization, wet type macular degeneration, rheumatoid arthritis,obesity or neurodegenerative disease in a patient comprisingadministering to a patient in need of such treatment a therapeuticallyeffective amount of the compound of claim 1.